Microgel compositions

ABSTRACT

This invention relates to microgel compositions, and in particular, to gel compositions formed by binding a plurality of individual microgel particles together. The present invention also relates to processes for the preparation of these compositions and their use for particular applications, especially medical applications such as the repair of damaged, degenerated or inappropriately formed load-bearing tissue (such as, for example, intervertebral discs).

This invention relates to microgel compositions, and in particular, togel compositions formed by binding a plurality of individual microgelparticles together. The present invention also relates to processes forthe preparation of these compositions and to their use for particularapplications, especially medical applications such as the repair ofdamaged, degenerated or inappropriately formed load-bearing tissue (suchas, for example, intervertebral discs).

BACKGROUND

Microgel particles, which are nanoscopic or microscopic colloidalparticles of cross-linked polymer, have been investigated for a numberof different potential applications. Particular examples include theiruse as micro-reactors for the template synthesis of inorganicnanoparticles, as optically active materials including lenses andphotonic crystals, and as drug delivery systems (Das et al. AnnualReviews of Materials Research, 2006, Vol. 36: 117-142).

Microgel particles have also been used for the preparation of photonichydrogels, especially photonic hydrogels capable of manipulating photonsin the visible and near-infrared spectrum (see Cai et al.Macromolecules, 2008, Vol. 41: 9508-9512). More specifically, Cai et al.describe photonic hydrogels derived from thermally-responsive, vinylfunctionalized microgel particles. The microgel particles, which areformed from PEG-polymers, are cross-linked by interlinking polymerchains formed by the polymerization of ethyleneglycolacrylate (PEGA)and/or acrylamide monomers. Upon photo-initiation, theethyleneglycolacrylate (PEGA) or acrylamide monomers react with thevinyl groups present on the microgel particles and polymerise to forminterlinking poly(PEGA) or poly(acrylamide) polymer chains. The resultis a hydrogel composed of microgel particles connected together byinterlinking polymer chains of varying length.

One particular application of biocompatible microgel particles is theirpotential utility for the replacement or repair of injured, degeneratedor inappropriately formed load-bearing soft tissues, such as, forexample, intervertebral discs and the tissues found in articular joints(such as the elbow, knee, hip, wrist, shoulder and ankle). These softtissues need to be able to bear significant loads and changes inpressure. For example, the pressures experienced within humanintervertebral discs can vary from about 0.5 MPa when sitting to about2.3 MPa when lifting a 20 kg weight. Consequently, the ability of softtissues, such as intervertebral discs, to bear varying biomechanicalloads is essential for the normal operation of the body.

The principle load-bearing tissue of the intervertebral disc is thedisc-shaped nucleus pulposus, which forms the centre of anintervertebral disc. The nucleus pulposus consists of chondrocytes(cartilage producing cells) within a matrix of collagen andproteoglycans. Articular cartilage, which is the tissue covering bonyends of articular joints, has a similar composition to that found in thenucleus pulposus. The proteoglycans have a high negative charge densityand are responsible for the high swelling pressure of the nucleuspulposus. The nucleus pulposus is a natural ionic hydrogel and containsabout 75% water in adults. The proteoglycan content gradually decreaseswith age due to natural degeneration, and this can result in theformation of three dimensional channels known as “clefts”. The formationof clefts provides weak points or voids in the structure of the disc,which can eventually become detrimental to the overall shape, form,dimensions and performance of the disc, particularly when a pressure isapplied.

Any injury, degeneration or malformation in load bearing tissues canresult in significant pain and lack of mobility. A major proportion ofall intervertebral discs in the lower part of the spine show signs ofdegeneration by the age of 50. This can result in chronic back pain,which is a major cause of morbidity and absence from work.

The treatment of damaged load-bearing soft tissues, such asintervertebral discs or articular joints, is usually directed atsymptomatic relief of the pain. In severe cases, surgical interventionmay be necessary to remove some of the damaged tissue and insert aprosthetic replacement. Surgical intervention is effective in relievingpain, but it can result in the damage of adjacent tissues andalterations in the biomechanical/load-bearing properties of the tissueconcerned. In addition, surgical intervention may require a protractedstay in hospital and significant morbidity for the patient concerned.

A material science approach to address the problem of degeneratingintervertebral discs and other load bearing tissues involves injectingmolecules that polymerise at the site of injection. The polymer depositformed provides additional mechanical strength to the bolster theremaining tissue. One particular example described in WO2000/062832 isthe in situ polymerisation of poly(ethylene glycol) tetra-acrylate inthe nucleus pulposus of the intervertebral disc. Another exampleinvolves the injection of chitosan into the nucleus pulposus andallowing it to polymerise. Chitosan is a positively chargedpolysaccharide that is soluble in water at low pH. It undergoes asolution-to-gel transition when the pH is increased. It has thereforebeen contemplated that chitosan may be injected as a low pH solution andthen allowed to form a gel when it is exposed to a higher pH in viva Thegel that forms in vivo is uncharged and forms a polymer network thatoccupies the whole volume of the injected solution. Hence, it becomes amacrogel through in situ polymerisation.

The provision of injectable materials that can be used to treat damagedor degenerated load-bearing tissues, such as intervertebral discs, is amajor challenge. It should also be appreciated that a key criterion forsuch materials is that their mechanical properties replicate that of thenormal healthy load-bearing tissue as closely as possible.

WO2007/060424, the entire contents of which are incorporated herein byreference, describes the use of pH-responsive microgel particles forthis particular application. The use of biocompatible pH-responsivemicrogel particles provides many attractions. In particular, themicrogel particles can be injected in a compacted (or “non-swollen”)configuration by controlling the pH of the injection medium. However,once present in the body, the pH will typically adjust to the normalphysiological pH of the tissue due to the natural buffers present inphysiological fluids. At physiological pH values, the polymer that formsthe pH-responsive microgel particles undergoes a conformational change,which causes the microgel particles to hydrate and swell. The swollenmicroparticles then provide a gelatinous mass which fills any regions ofdegenerated tissue and provides additional mechanical support to thetissue concerned.

However, despite the attractions of this approach, the mechanicalproperties of the gel is not optimal and there is a tendency for themicrogel particles to dissipate/migrate away from the injection site, sothere still remains a need for alternative injectables that are capableof providing further improved biomechanical support for the treatment orreplacement of damaged or degenerated load-bearing tissues.

It is therefore an object of the present invention to obviate ormitigate one or more of the problems of the prior art, whetheridentified herein or elsewhere. In particular, it is an object of thepresent invention to provide a further improved method for repairingdamaged and degenerated load-bearing tissue.

BRIEF SUMMARY OF THE DISCLOSURE

The present invention provides novel microgel compositions havingimproved mechanical properties that enable them to be used for therepair and/or replacement of damage soft tissue, such as intervertebraldiscs, as well as for other applications.

In its broadest terms, the present invention provides a compositioncomprising a plurality of microgel particles, wherein adjacent microgelparticles are bound together by either

-   -   (i) covalent cross-links formed by the reaction of        vinyl-containing moieties grafted onto the surfaces of the        microgel particles; and/or    -   (ii) by a cross-linked polymer network that interpenetrates        adjacent microgel particles and thereby binds the particles        together, wherein the vinyl polymer network is formed by the        polymerisation of a water soluble cross-linking monomer        comprising two or more vinyl groups.

The compositions of the present invention possess advantageousmechanical properties, particularly in terms of their ability to supportloads. The mechanical properties of these compositions can be readilyand advantageously fine tuned and controlled in order to optimise themechanical properties of the resultant hydrogel composition forload-bearing applications. The mechanical properties can be altered by,for example, modifying the preparation conditions of the composition,varying the parent microgel particles, modifying the cross-linkingreaction conditions, altering the pH of the composition or theconcentration of the microgel particles.

In particular, the mechanical properties of the compositions of theinvention can substantially replicate those of normal healthyload-bearing tissue, such as, for example, intervertebral discs, andthus allow for the provision of hydrogel compositions materials that canbe used to treat damaged or degenerated load-bearing tissues. Moreover,the compositions of the invention can advantageously be formed in situat the desired target site. This enables the precursor materials(including, inter alia, the microgel particles, cross-linking monomersand/or other required reactants) to be administered in a convenient form(e.g. by liquid injection) to a target site (e.g. in vivo) before thehydrogel compositions and/or doubly cross-linked (DX) microgels are dulyformed and molded in situ within the desired target site.

Compositions of the present invention also provide for a more consistentphysical form, which is particularly advantageous for in vivoapplications where predictability of the final form is crucial. Suchcompositions are also stable and robust, having high critical strains,and have a low propensity to migrate or redisperse when serving aload-supporting function, especially in vivo. In particular, suchcompositions have a reduced propensity to redisperse in aqueous alkalineor acidic environments.

The compositions of the present invention have the further advantagethat they can be formed from their conveniently administrable precursorsusing either temperature of pH-triggered swelling of the microgelparticles. This enables the precursor components required to form thecompositions of the invention to be effectively stored for long periodsbefore administration and composition formation. This is particularlyadvantageous for medical applications where the administrable form ofthe composition must satisfy regulations and requirements formanufacture, transport, and storage of the administrable form. Moreover,for in vivo applications, the properties of the compositions can bemodified to allow a physiological pH to provide the pH-triggeredswelling of the microgel particles. For example, physiologicalpH-triggered swelling of the microgel particles can cause adjacentmicrogel particles to enlarge and inter-penetrate with theirneighbouring precursor microgel particles and facilitates thecross-linking of adjacent microgel particles to form the compositions ofthe invention. This is advantageous because particles maintain a3-dimensional connected (non-porous) structure with maximised loaddistribution within the gel.

Advantages of the DX microgels over singly cross-linked (SX) microgelsinclude higher elastic modulus values, higher yield strains, andswelling in aqueous solutions without any re-dispersion.

Although DX microgels resemble a hydrogel in that they are macroscopic,they are also very different because DX microgels are composed ofinter-linked nanometer (or sometimes micrometer)-sized microgelparticles. This means that there mechanical and swelling properties canbe altered at the size scale of the microgel particles. This offers newpossibilities to tune the mechanical properties, construct hybrids andblends, that do not exist for conventional macromolecular hydrogels. Theterm macromolecular hydrogel refers to hydrogels formed by covalentlylinking molecules—conventional hydrogels.

A particular advantage of the DX microgel preparation methods of thepresent invention is that the microgel particles inter-penetrate priorto double crosslinking. That means that there is an efficient,three-dimenstional, network in place for distributing stress once thematerial is formed. As such, modulus values are higher than those of theprecursor SX physical gels.

Thus, in a particular aspect, the present invention provides acomposition comprising a plurality of microgel particles, whereinadjacent microgel particles are covalently bound together by covalentcross-links formed by the reaction of vinyl-containing moieties graftedonto the surfaces of the microgel particles. In this aspect of theinvention, vinyl functionalized microgel particles are directlycross-linked to each other without any intervening cross-linker(s). Thiscan be advantageous because the cross-linking chemistry is simple andrequires fewer reagents. The resultant hydrogel compositions alsopossess a more consistent physical form, which is generally more robustwith a lower propensity for migration and/or re-dispersement whensupporting loads (e.g. in vivo). Moreover, administration ofcompositions of the present invention is particularly convenient(especially when forming the composition in vivo). These compositionsalso possess advantageous mechanical properties, particularlyadvantageous elastic properties. These compositions also displayexcellent gel rheology with low viscosity, making them ideal for softtissue repair. Moreover, the mechanical properties of the compositionscan be readily fine tuned and controlled by merely altering the degreeof vinyl functionalisation of the microgel particles, and also theconcentration of the microgel particles used during compositionpreparation. As such, compositions can be tailored for a variety ofspecific applications.

In a further aspect the present invention provides a process for thepreparation of a composition comprising a plurality of microgelparticles, wherein adjacent microgel particles are bound together bycovalent cross-links formed by the reaction of vinyl-containing moietiesgrafted onto the surfaces of the microgel particles, the processcomprising:

-   -   (i) providing, in an aqueous medium, a plurality of microgel        particles comprising functional vinyl-containing moieties        grafted onto the surfaces of the microgel particles; and    -   (ii) causing the microgel particles to swell so that adjacent        microgel particles are brought into contact with one another and        facilitating the free radical coupling of the vinyl groups to        covalently bind adjacent microgel particles together.

In a further aspect the present invention provides a microgel particlecomprising a plurality of vinyl-containing moieties grafted onto thesurface of the microgel particle.

In a further aspect the present invention provides a process ofpreparing a microgel particle comprising a plurality of vinyl-containingmoieties grafted onto the surface of the microgel particle, the processcomprising reacting a microgel particle with a compound of the formula:

Z-L-B

wherein Z, L and B are as defined herein.

In a further particular aspect, the present invention provides acomposition comprising a plurality of microgel particles that are boundtogether by a cross-linked polymer network that interpenetrates adjacentmicrogel particles, wherein the vinyl polymer network is formed by thepolymerisation of a water soluble cross-linking monomer comprising twoor more vinyl groups. The compositions of this particular aspect of theinvention have the advantage that compositions with highly desirablemechanical properties, particularly those suitable for soft tissuerepair, can be formed without the need to pre-functionalise the microgelparticles with vinyl-containing moieties. This can simplify thecomposition formation process for certain applications, and potentiallysimplifies the manufacture of the administrable form of thecompositions, especially where in vivo applications are intended.Moreover, such compositions still allow for temperature and/orpH-triggered swelling, the advantages of which are outlined above,particularly with respect to in vivo applications. The properties ofsuch compositions can also be readily controlled and fine tuned byvarying the molecular weight of the cross-linking monomer.

In an embodiment, the composition is substantially free of any directcross-linking between the microgel particles (i.e. there are no directcovalent cross-links formed between the polymer chains making upadjacent microgel particles).

Alternatively, the compositions of this aspect of the invention mayfurther comprise covalent cross-links formed by the reaction ofvinyl-containing moieties grafted onto the surfaces of adjacent microgelparticles. These covalent cross-links may suitably be in addition to thecross-linked polymer network that interpenetrates adjacent microgelparticles. As such, the composition may comprise microgels that arebound by both direct cross-linking and a separately interpenetratingpolymer network.

Alternatively or additionally, the composition may comprise a degree ofindirect cross-linking between microgel particles, for instance, wherethe microgel particles pre-functionalised with cross-linkable vinylmoieties are cross-linked via the cross-linking monomer (i.e. one of thevinyl groups of the cross-linking monomer reacts with a vinyl group onone microgel particle whilst another of the vinyl groups of thecross-linking monomer reacts with a vinyl group on another microgelparticle). The use of a cross-linking monomer comprising two or morevinyl groups, e.g. a bi-vinyl cross-linking monomer, is advantageousover the use of cross-linking monomers comprising only a single vinylgroup because the chain length of any indirect cross-links are generallybetter regulated, thus the composition's properties are more easilycontrolled and fine tuned through selection of the appropriatecross-linking monomer. Where, as in the case Cai et al. (Macromolecules,2008, Vol. 41: 9508-9512), a cross-linking monomer comprises only asingle vinyl group, cross-links between microgels are formed withvarying chain lengths (following the propagated polymerization of thecross-linking monomers themselves), which effects control of theproperties of the resulting composition.

In a further aspect, the present invention provides a process for thepreparation of a composition comprising a plurality of microgelparticles that are bound together by a cross-linked polymer network thatinterpenetrates adjacent microgel particles and thereby binds theparticles together, wherein the vinyl polymer network is formed by thepolymerisation of a water soluble cross-linking monomer comprising twoor more vinyl groups, the process comprising:

-   -   (i) providing, in an aqueous medium, a plurality of microgel        particles; and    -   (ii) causing the microgel particles to swell in the presence of        a water soluble cross-linking monomer comprising two or more        vinyl groups such that adjacent microgel particles are brought        into contact with one another and facilitating the        polymerisation of the cross-linking monomer to form a        cross-linked polymer network that interpenetrates the particles        and binds adjacent microgel particles together.

In a further particular aspect, the present invention provides acomposition comprising a plurality of microgel particles, whereinadjacent microgel particles are bound together by a combination of:

-   -   (i) covalent cross-links formed by the reaction of        vinyl-containing moieties grafted onto the surfaces of the        microgel particles; and    -   (ii) by a cross-linked polymer network that interpenetrates        adjacent microgel particles and thereby binds the particles        together, wherein the vinyl polymer network is formed by the        polymerisation of a water soluble cross-linking monomer        comprising two or more vinyl groups.

In a further aspect the present invention provides a compositionobtainable by any one of the processes defined herein.

In a further aspect, the present invention provides a precursorcomposition for forming a composition of the invention as definedherein, the precursor composition comprising a plurality of microgelparticles together with one or more additional cross-linking reactantsor reagents (e.g. cross-linking monomers, vinyl polymerisationinitiators etc.). Suitably, the microgel particles are in a non-swollenconfiguration in the precursor composition, thereby enabling them to beconveniently stored and administered to the target site for in situformation of the composition of the invention.

In a further aspect, the present invention provides a method of treatinga subject suffering from a condition characterised by damaged ordegenerated soft tissue, the method comprising administering to asubject in need of such treatment, a therapeutically effective amount ofa composition as defined herein.

In a further aspect, the present invention provides a composition asdefined herein for use in the treatment of a condition characterised bydamaged or degenerated soft tissue.

In a further aspect, the present invention provides a method of treatinga subject suffering from a condition characterised by damaged ordegenerated soft tissue, the method comprising forming a composition asdefined herein in situ within the body.

In a further aspect, the present invention provides a composition asdefined herein for use in the treatment of a condition characterised bydamaged or degenerated soft tissue, wherein said composition is formedin situ within the body.

In a further aspect, the present invention provides a method of treatinga subject suffering from a condition characterised by damaged ordegenerated soft tissue, the method comprising administering a precursorcomposition as defined herein which reacts to form a composition asdefined herein in situ within the body.

In a further aspect, the present invention provides a precursorcomposition as defined herein for use in the treatment of a conditioncharacterised by damaged or degenerated soft tissue, wherein saidcomposition forms a composition of the invention as defined herein insitu within the body.

The above and further aspects of the invention are described in furtherdetail herein.

BRIEF DESCRIPTION OF THE DRAWINGS

Particular embodiments of the invention are further describedhereinafter with reference to the accompanying drawings, in which:

FIG. 1 is a scheme showing a first method for preparing a cross-linkedmicrogel particle composition of the present invention;

FIG. 2 is a scheme showing a second method for preparing a cross-linkedmicrogel particle composition of the present invention;

FIG. 3 is a scheme showing a third method for preparing a cross-linkedmicrogel particle composition of the present invention; and

FIGS. 4 to 18 are described in the accompanying examples.

DETAILED DESCRIPTION Microgel Compositions

The present invention provides microgel compositions that possessparticularly advantageous mechanical properties that render themsuitable for a number of applications, including the repair of damagedor degenerated soft tissue.

The compositions of the invention comprise microgel particles that areeither bound together by covalent cross-links formed by the reaction ofvinyl containing moieties grafted onto the surface of the microgelparticles; and/or by a cross-linked polymer network that interpenetratesadjacent microgel particles and thereby binds the particles together,wherein the polymer network is formed by the polymerisation of a watersoluble cross-linking monomer comprising two or more vinyl groups.

In a further aspect, the present invention provides a precursorcomposition for forming a composition of the invention as definedherein, the precursor composition comprising a plurality of microgelparticles together with one or more additional cross-linking reactantsor reagents (e.g. cross-linking monomers, vinyl polymerisationinitiators etc.). Suitably, the microgel particles are in a non-swollenconfiguration in the precursor composition, thereby enabling them to beconveniently stored and administered to the target site for in situformation of the composition of the invention.

The present invention further provides a composition obtainable by anyone of the processes defined herein.

In addition, the present invention further provides a compositionobtained by any one of the processes defined herein.

The present invention also provides a composition directly obtained byany one of the processes defined herein.

As discussed in further detail below, the approaches to bind themicrogel particles together rely on free-radical chemistry to induce thecoupling of vinyl groups (whether it is the free radical polymerisationof the cross-linking monomers comprising two or more vinyl groups or thevinyl containing moieties grafted onto the surface of the microgelparticles, or a combination thereof).

Microgel Particles

The compositions of the present invention are macrogel hydrogelcompositions that are formed by binding together a plurality of microgelparticles.

By the term “microgel particle”, we mean a hydrogel particle having asize within the range of 1 nm to 100 μm and which comprises across-linked polymer formed by the polymerisation of a plurality ofcross-linked co-monomers.

The microgel particle itself may be considered as being onemacromolecule (i.e. the cross-linked polymer) comprising a molar mass ofbetween about 10⁶ and 10¹⁰ Da. (e.g. between 10⁶ and 10⁹ Da). However,the individual co-monomers that were used during the preparation of themicrogel particles may comprise a molar mass of between about 5Da and5,000 Da, more preferably, between about 10 Da and 1,000 Da, even morepreferably, between about 50 Da and 500 Da, and most preferably, betweenabout 75 Da and 400 Da. In a most preferred embodiment, the co-monomersused in the polymerisation reaction comprise a molar mass of betweenabout 100 Da and 300 Da.

The microgel particle is suitably a cross-linked co-polymer particlethat is pH and/or temperature responsive. By “pH and/or temperatureresponsive” we mean that the polymer that forms the microgel particlescan undergo a pH and/or temperature dependent conformational change,which has a consequential effect on the hydration of the particle. Thismeans that by varying the pH and/or temperature, the microgel particlescan transition between a collapsed configuration, in which the particleis in a compact configuration, to a swollen configuration in which theparticle is in the form of a highly hydrated gel (or microgel).

By the term “collapsed configuration”, we mean the particle issubstantially reduced in size and has a smaller average diameter than inthe swollen configuration. In this state, the polymer present in themicrogel particles adopts a configuration which does not favour theingress of water into the particle. The limit of the collapsedconfiguration is when the particle contains virtually no water. Hence,the microgel particle preferably comprises less than about 70% (w/w)water, more preferably, less than about 50% (w/w) water, preferably,less than about 30% (w/w) water, and even more preferably, less thanabout 20% (w/w) water, and most preferably, less than about 10% (w/w)water in the collapsed configuration. In a particular embodiment, theparticles comprise a minor proportion of water (less than about 40% w/w)in the collapsed configuration. It will be appreciated that this watercontent is a reference to the water present within the particle.

It will be appreciated that the diameter of the microgel particles willdepend upon the hydration (water content) thereof which is in turndependent upon the configuration of the polymer. The diameter of themicrogel particle in the collapsed configuration is typically less thanabout 100 μm, more typically, less than about 50 μm, and even moretypically, less than about 20 μm. However, in a preferred embodiment, itis preferred that the diameter of the microgel particle in the collapsedconfiguration is less than about 10 μm, more preferably, less than about5 μm, and even more preferably less than about 1 μm. Most preferredparticles are on the nanometre scale, i.e. the average diameter of themicrogel particle in the collapsed configuration is preferably betweenabout 1 nm and 1000 nm, more preferably, between about 10 nm and 750 nm,even more preferably, between about 20-500 nm, and most preferably,between about 50 and 100 nm in diameter.

By the term “swollen configuration”, we mean the microgel particle issubstantially hydrated and enlarged, and therefore has a greater averagediameter than the when the particle is in the collapsed configuration.It will be appreciated that this swelling is caused by a flow of waterinto the particle. In the swollen configuration, the microgel particlepreferably comprises at least about 70% (w/w) water, more preferably, atleast about 85% (w/w) water, preferably, at least about 90% (w/w) water,even more preferably, at least about 95% (w/w) water, and mostpreferably, at least about 99% (w/w) water. It will be appreciated thatthe amount of water in the particle will depend on the temperatureand/or pH as well as the properties of the polymer making up themicrogel particle (e.g. charge density). Suitably, the average diameterof the microgel particle is adapted to increase by at least 20%, moresuitably, by at least 50%, more suitably by at least 100%, even moresuitably, by at least 200% as it transitions from a collapsed to aswollen configuration in response to a change in the pH and/ortemperature.

In an embodiment, the diameter of the microgel particles in the swollenconfiguration is about 5 nm to 100 μm, suitably about 5 nm to 10 μm, andpreferably 50 nm to 1 μm.

For concentrated dispersions of microgel particles (e.g. concentrationsgreater than 2 wt. %), the transition from a collapsed configuration toa swollen configuration can be referred to as a pH or temperaturedependent macrogelation step. The conformational change of the polymercauses solvent in the surrounding medium to ingress into the particleand cause it to swell. Thus, in the collapsed configuration theparticles are dispersed in a substantially fluid medium, which has a lowviscosity and can flow. Thus, in this configuration, the microgelparticles can be easily transported to the desired location, forexample, by injecting the particles to the desired location in the body.However, in the swollen configuration, the microgel particles form agelled mass having a higher viscosity and a physical gel of higherviscosity.

For the particular application whereby the compositions of the inventionare used for the treatment of damaged or degenerated load-bearingtissue, the polymer that makes up the microgel particle can be selectedso that it transitions to a swollen configuration at physiological pH ortemperature. This means that the pH or temperature of the injectionmedium can be manipulated so that the microgel particles are in theircompact configuration at the point of administration, thereby enablingthem to be easily administered to the desired location by injection. Thesubsequent change in temperature and/or pH in the body will then causethe microgel particles to swell so that they contact adjacent microgelparticles. The swollen microgel particles can then be bound together bythe reaction between the vinyl groups provided on or proximate to thesurface of the adjacent swollen microgel particles and/or by theformation of a cross-linked polymer network within the swollen microgelmatrix. The result is a cohesive macrogel composition havingadvantageous mechanical properties.

In order for the microgel particles to swell, they need to be dispersedin a suitable aqueous medium. Water, buffer or physiological fluids arepreferred.

The plurality of microgel particles used to form the compositions of thepresent invention may all possess the same polymeric composition, i.e.the same co-monomers are used to form the polymers that make up themicrogel particles. However, in certain embodiments of the invention,the plurality of microgel particles may comprise two or more differenttypes of microgel particle formed from polymers that are made up ofdifferent co-monomeric components or with different ratios of the sameco-monomeric components.

A microgel dispersion is different to a hydrogel because it has theability to flow and exist in the fluid state. A hydrogel cannot do thatbecause it is a macroscopic (e.g., millimetre or centimetre sizedmaterial). The microgel dispersion consists of microgel particlesdispersed within an (aqueous) solution. Because there is space betweenthe particles they can flow and it is a fluid. However, using thepH-responsive microgel particles of the present invention, pH is used totrigger an increase in the size of the microgel particle so that theyoccupy the whole volume of the fluid. This causes formation of a (singlycrosslinked) physical gel. In this state the peripheries of the microgelparticles inter-penetrate.

The new method for DX microgel formation takes advantage of this bycovalently coupling the peripheries of inter-penetratingvinyl-functionalised microgels. This gives a second level ofcrosslinking (double crosslinked) that links the microgel particlestogether.

pH-Responsive Microgel Particles

In a particular embodiment, the microgel particles are pH responsive.Any suitable pH-responsive microgel particles may be used to form thecompositions of the present invention.

In a particular embodiment, the pH-responsive polymer is a polymerdefined in WO2007/060424, the entire contents of which are incorporatedherein by reference. In particular, suitable polymers for forming pHresponsive microgel particles are defined at page 12/line 21 to page22/line 17, and page 26/line 20 to page 28/line 22, of WO2007/060424.The microgel particles may be made by any suitable methods known in theart. Suitable initiators to use in the formation of such pH-responsivemicrogel particles are defined at page 22/line 19 to page 24/line 3 ofWO2007/060424. Suitable surfactants that may also be used are defined atpage 24/line 5 through to page 26/line 11.

For the compositions of the present invention, it is preferred that themicrogel particle comprises a hydrophobic co-monomer. Hence, it ispreferred that the microgel particle comprises a co-polymerised polymerparticle, which may be defined by the following formula I:

Poly(B-co-P-co-X)  (I)

wherein:

-   -   P is a pH-responsive co-monomer;    -   X is a functional cross-linking co-monomer; and    -   B is a hydrophobic co-monomer.        These particular microgel polymers are described at page 19/line        21 through to page 22/line 8 of WO2007/060424, the relevant        contents of which are incorporated herein by reference.

In a preferred embodiment, the microgel particle comprises ethylacrylate(i.e. EA, which is the hydrophobic co-monomer, B), methacrylic acid(i.e. MAA, which is the pH responsive co-monomer, P), and 1,4-butanedioldiacrylate (i.e. BDDA, which is the functional cross-linking co-monomer,X). Accordingly, a preferred microgel particle comprisespoly(EA/MAA/BDDA).

The poly(EA/MAA/BDDA) used to form the microgel particle may comprise amaximum mass % EA (hydrophobic monomer) of about 95%, a minimum mass %MAA (pH-responsive monomer) of about 5%, and a minimum mass % BDDA(cross-linking monomer) of about 0.1%. Suitably the mass % of BDDA iswithin the range of 0.1 to 2%.

In a particular embodiment, the poly(EA/MAA/BDDA) microgel particlescomprise about 65.9% EA, about 33.1% MAA and about 1.0% BDDA based onthe total monomer mass. This may be defined as a mass ratio ofEA/MAA/BDDA as 65.9/33.1/1.0, or as a mole ratio of EA/MAA/BDDA is130.4/76.0/1.0.

In another preferred embodiment, the microgel particle comprisesmethylmethacrylate (i.e., MMA, which is the hydrophobic co-monomer, B),methacrylic acid (i.e., MAA, which is the pH-responsive co-monomer, P)and ethyleneglycol dimethacrylate (i.e., EGDMA, which is the functionalcross-linking co-monomer, X).

Accordingly another preferred microgel particle comprisespoly(MMA/MAA/EGDMA).

The poly(MMA/MAA/EGDMA) used to form the microgel particle may comprisea maximum mass % MMA (hydrophobic monomer) of about 95%, a minimum mass% MAA (pH-responsive monomer) of about 5%, and a minimum mass % EGDMA(cross-linking monomer) of about 0.1%. Suitably the mass % of EGDMA iswithin the range of 0.1 to 2%.

In a particular embodiment, the poly(MMA/MAA/EGDMA) of the microgelparticles comprises about 66.8% MMA, about 32.8% MAA and about 0.4%EGDMA based on the total monomer mass. This may be defined as a massratio of MMA/MAA/EGDMA of 167/82/1.0, or as a mole ratio ofMMA/MAA/EGDMA is 320/185/1.0.

In a particular embodiment, the composition or precursor composition asdefined herein comprises microgel particles which swell or collapse as aconsequence of a change in the pH of the surrounding environment. In aparticular embodiment, the composition or precursor composition has astorage state and/or administration state having a pH environment whichis different from the pH environment of the target site (e.g.physiological pH). In the storage or administration state, the microgelparticles suitably exist in a substantially non-swollen state. Thedifference between the pH environment of the storage and/oradministration state and the pH environment of the target site issuitably sufficient to cause the microgel particles to swell such thattheir hydrodynamic diameter (d_(h)) increases. Suitably, the pH at thetarget site causes the hydrodynamic diameter (d_(h)) of the microgelparticles to increase relative to the storage or administration state byat least 10%, more suitably by at least 25%, even more suitably by atleast 50%, and most suitably by at least 100%. The target site maysuitably be in vivo, having a physiological pH environment.

Suitable pH-responsive microgel particles can be sourced commercially orprepared using methodology well known in the art.

Temperature-Responsive Microgel Particles

The microgel particles of the present invention may also be temperatureresponsive. Any suitable temperature-responsive microgel particles maybe used to form the compositions of the present invention.

The term “temperature-responsive” is used herein to refer to polymersthat undergo a temperature dependent change in hydration. Thetemperature at which a substantial change in polymeric hydration occursis known as the critical solution temperature (CST). The lower criticalsolution temperature (LOST) is the critical temperature below which theco-polymer becomes highly miscible with water. Accordingly, above theLOST the co-polymer is highly dehydrated and below the LOST theco-polymer is highly hydrated. Suitable polymers of the presentinvention have an LOST within the range of 20° C. to 40° C. Thedesirable LOST will be dictated ultimately by the intended applicationof the microgel composition. For example, for in vivo application, itwill be desirable to have a LOST above 37° C. For other applications,for example the provision of a temperature responsive microgel film forcell culture applications, a LCST of, for example, 30 to 34° C. may berequired. An example of a polymer having a lower LCST of approximately32° C. is Poly(N-isopropylacrylamide). The term “temperature-responsive”is also used herein to refer to monomers which, when polymerised, formtemperature-responsive polymers that undergo a temperature dependentchange in hydration as discussed above.

In a particular embodiment of the invention, the temperature responsivepolymer used to form the microgel particles is a co-polymer of thefollowing formula II:

Poly(C-co-Q-co-X)  (II)

wherein:

-   -   C is a temperature responsive monomer;    -   Q is a monomer containing a hydroxyl group or a pH-responsive        co-monomer P as defined hereinbefore; and    -   X is a cross-linking co-monomer as defined in WO2007/060424.

Any suitable temperature-responsive monomer (component C) may be used.Suitable examples of the temperature-responsive monomer C includeN-isopropylacrylamide and vinylcaprolactone. Suitably, C constitutes 40to 98 mol. % of the temperature responsive polymer.

A suitable example of Q is hydroxy ethyl methacrylate, vinyl alcohol,ethylene glycol methacrylate, or poly(ethylene glycol) methacrylate.Suitably, C constitutes 1 to 55 mol. % of the temperature responsivepolymer.

As above, X suitably constitutes 0.01 to 2 mol. % of thetemperature-responsive polymer.

The polymers used to prepare the microgel particles of the presentinvention may further comprise a monomer comprising a vinyl-containingside chain (in place of, or in addition to, the cross-linking monomerX). The vinyl containing side chain provides functional vinyl groups, atleast a proportion of which will be on or proximate to the surface ofthe microgel particle and will therefore provide a means by which themicrogel particles can react and bind to one another. A suitable exampleof such a monomer would be allyl methacrylate (AM). A suitable exampleof a polymer comprising allyl methacrylate is poly(EA/MAA/AM) as definedin Dalmont et al. (Langmuir, 2008, 24, 2834-2840).

Grafting of Vinyl-Containing Moieties

In a particular embodiment, the present invention provides a compositioncomprising a plurality of microgel particles, wherein adjacent microgelparticles are covalently bound together by cross-linking groups formedby the reaction of vinyl-containing moieties grafted onto the microgelparticles. As such, the precursor composition may suitably comprisemicrogel particles with vinyl-containing moieties grafted thereon.

It is predicted that the vinyl-containing moieties will be predominantlygrafted on to the surface of the microgel particle, or proximatethereto.

The grafting of vinyl-containing moieties onto the pre-formed microgelparticles provides a plurality of vinyl-containing moieties that can besubsequently reacted to form covalent cross-links between adjacentmicrogel particles.

Any suitable vinyl-containing moiety that can be grafted onto themicrogel particle may be used for this purpose. Suitably, thevinyl-containing moiety is water soluble.

In an embodiment, the vinyl containing moiety is provided by reacting amicrogel particle with a water soluble compound of the formula (III):

Z-L-B  (III)

wherein:

-   -   Z is a reactive group;    -   L is a bond or linking group between Z and B; and    -   B is a group comprising a vinyl functional group.

Z may be any suitable reactive group. The purpose of the group Z is toreact with a functional group present on the microgel particle andthereby graft the -L-B portion of the compound of formula III onto theparticle surface to provide the vinyl-containing moiety. Thus, theselection of a suitable functional group Z will be dictated by thenature of the microgel particle concerned. A skilled chemist would bereadily able to select suitable groups. For example, if the microgelparticle comprises a carboxylic acid groups, then Z could be any groupthat will react to form an ester with the carboxylic acid group, suchas, for example, a halogen, hydroxyl, amino or an epoxide group.Similarly, if the microgel particle comprises an amino group, then Zcould be a group that reacts with the amine to form an amide bond (forexample, Z could be a group —C(O)M, where M is a leaving group, e.g. ahalogen such as chloro), or a group that reacts to form a sulfonamidelinkage (e.g. Z is a group such as —S(O)₂Cl).

Alternatively, if the microgel particle comprises a carboxylic acidgroup (or groups), Z may suitably be an amino group, such as aminoethylmethacrylate (or salt thereof), and coupling may suitably lead toan acid amide. In such cases, compound III may suitably be coupled tothe carboxylic acid group(s) following preactivation of the carboxylicacid group(s) (e.g. via the formation of an acyl-chloride) or using acoupling agent (e.g. 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide(EDC), carbonyldiimidazole (CDI)). Z may suitably be alkylamino (or asalt thereof), for example ethylamine hydrochloride.

In a particular embodiment, Z is an epoxide group.

L may be bond or any suitable linker group, such as, for example, afunctionalised alkylene chain optionally comprising one or morefunctional groups selected from —O—, —O(O)—, —O(O)O—, —OC(O)—, —NR^(a)—,—NR^(a)—C(O)—, or —C(O)—NR^(a)—, wherein R^(a) is H or (1-2C)alkyl or Lmay be —(OCH₂CH₂)_(n), where n is 1 to 50 (inclusive). The alkylenechain may be a short (1-3 carbon atom) group comprising one or more ofthese functional groups defined above.

B may be any suitable vinyl-containing group. In a particularembodiment, B is a group —CR₁═CR₂R₃, i.e. the vinyl-containing moiety isa compound of structural formula IV shown below:

wherein Z and L are as defined above; and R₁, R₂ and R₃ are selectedfrom H or (1-3C)alkyl.

R₁, R₂ and R₃ are suitably selected from H, methyl or ethyl, especiallyH or methyl.

In an embodiment of the invention, the compound of formula III or IV isselected from glycidyl methacrylate, glycidylacrylate or otherfunctionalised glycyidylacrylates. Such compounds can be coupled tocarboxylic acid, amine or hydroxyl groups on the microgel particlesurface.

In a particular embodiment, the compound of formula III or IV isglycidyl methacrylate.

In an alternative embodiment, the compound of formula III or IV isallylamine, which can be covalently linked to carboxylic acid groups onthe microgel particle surface using water-soluble carbodiimidechemistry.

In an alternative embodiment, the compound of formula III or IV isaminoethyl methacrylate hydrochloride (AEMHCI). Such compounds maysuitably be coupled to a carboxylic acid group(s) upon the microgelparticles through a coupling reaction involving EDC.

A person skilled in the art will be able to select suitable experimentalconditions to graft the vinyl-containing moiety onto the microgelparticle.

Suitably, the reaction will be carried out in the aqueous solvent at apH of between 2 and 7, and preferably at a pH of between 2 and 4.

Suitably, the concentration of the microgel particles in the aqueoussolvent is between 0.05 and 20 wt. %.

Suitably, the concentration of the vinyl-containing group (e.g. glycidylmethacrylate, glycidylacrylate or other functionalisedglycyidylacrylates) is between 10⁻³ and 10 Mol dm⁻³, and preferablybetween 0.1 and 5 Mol dm⁻³.

A suitable temperature for the grafting reaction is between 0 and 100°C., and preferably between 40 and 70° C. The reaction may proceed forbetween 0.5 and 48 hours, and preferably between 4 and 12 hours.

Suitably the vinyl-containing moiety should correspond to aconcentration of between 0.1 and 60 mol. % with respect to all of theco-monomers present in the gel. Preferably, the concentration of thevinyl-containing moiety should be between 10 and 30 mol. % with respectto all of the co-monomers present in the gel.

Cross-Linking of the Vinyl-Grafted Microgel Particles

The vinyl-containing moieties grafted onto the surface of the microgelparticles may undergo a free-radical coupling reaction directly with thevinyl-containing moieties grafted onto the surfaces of adjacent microgelparticles to form a direct covalent bond therebetween.

This particular reaction is shown schematically in FIG. 2. The firststep of the reaction involves providing microgel particles that havevinyl-containing moieties grafted on to their surfaces. The next stepinvolves bringing the surfaces of the adjacent particles into contactwith one another. This can be achieved by causing the responsivemicrogel particles to swell by varying the temperature or pH (asdescribed hereinbefore). The swelling of the microgel particles as theyhydrate causes the surfaces of adjacent particles to contact one anotherand even overlap to form interpenetrating regions of gelled polymer.This disposes the surface grafted vinyl-containing moieties of adjacentmicroparticles in close proximity to one another to facilitate thefree-radical coupling of the vinyl moieties, as discussed further below.

The reaction between the vinyl-containing moieties grafted onto thesurface of adjacent microgel particles is achieved by free-radicalchemistry using techniques well known in the art. A key feature of thepresent invention is that the reaction must take place in the aqueousmedium, so suitably water soluble reactants need to be used. For in vivoapplications it is also preferred that the any reactants used possesslittle or no toxicity to the subject.

Suitably, the reaction is conducted in the presence of a free radicalinitiator (hereinafter referred to as an initiator), which is watersoluble. Suitably, the initiator is responsive to temperature orultraviolet radiation.

Suitable water soluble initiators include:

Anionic Initiators:

-   -   initiators of the general formula [M]S₂O₈ ²⁻, wherein M is a        cation such as K⁺, Na⁺ or NH₄ ⁺, or a divalent cation. Ammonium        persulfate, (NH₄ ⁺)₂S₂O₈ ²⁻, is a specific example.    -   an organic anionic azo initiator of formula V:

[R⁹⁰R⁹¹(CN)C—N═N—(CN)R⁹²R⁹³]  (V)

-   -   wherein:        -   R⁹⁰ and R⁹² may be independently selected from a group            consisting of H; CH₃; a linear or branched (1-10C)alkyl            group; or a —NH—(1-10C)alkyl or —N[(1-10C)alkyl]₂ group; and        -   R⁹¹ and R⁹³ may be CR⁹⁴COOH (wherein R⁹⁴ may be —CH₂—,            —CH₂CH₂— or a linear, or branched (1-20C)alkylene chain) or            phenyl which is optionally substituted (for example, by one            to three substituent groups selected from halo, (1-6C)alkyl,            amido, amino, hydroxy, nitro, and (1-6C)alkoxy).

A particularly suitable initiator belonging to this group isazobiscyanopentanoic acid (also known as 4,4′-azobis(4-cyanovalericacid)).

Cationic initiators:

-   -   a cationic amine initiator of structural formula VI:

[R⁸⁰R⁸¹R⁸²C—N═N—R⁸³R⁸⁴R⁸⁵]xHCl  (VI)

-   -   wherein R⁸⁰, R⁸¹, R⁸³ and R⁸⁴ may be independently selected from        a group consisting of H; CH₃; a linear or branched (1-10C)alkyl        group; a —NH—(1-10C)alkyl or —N[(1-10C)alkyl]₂ group; and        wherein R⁸² and R⁸⁵ may be C(═NR⁸⁶)NH₂ wherein R⁸⁶ may be        independently selected from a group consisting of H; CH₃; a        linear or branched (1-10C)alkyl group.

For example, a specific example is propanimidamide,2,2′-azobis[2-methyl-, dihydrochloride]. This initiator is also known asV50.

Peroxide Initiators:

-   -   a peroxide initiator defined by the structural formula VII:

R⁷⁰—O—O—R⁷¹  (VII)

-   -   wherein R⁷⁰ or R⁷¹ may be independently selected from a group        consisting of H; CH₃; a linear or branched (1-10C)alkyl group; a        —NH-(1-10C)alkyl or —N[(1-10C)alkyl]₂ group; or phenyl which is        optionally substituted (for example, by one to three substituent        groups selected from halo, (1-6C)alkyl, amido, amino, hydroxy,        nitro, and (1-6C)alkoxy).

Suitable water soluble ultraviolet photoinitiators are of the formulaVIII:

R⁵²-ph-R⁵³  (VIII)

where R⁵² is HO—(CH₂)₂— and R⁵³ is —C(O)C(OH)(CH₃)₂ and ph represents aphenyl ring.

A particular initiator according to this formula is known as Irgacure2959.

The free-radical coupling reaction may also be conducted in the presenceof a suitable water soluble accelerator. Suitable examples of suchaccelerators include TEMED (1,2-bis(dimethylamino)ethane,N,N,N′,N′-Tetramethylethylenediamine) and ascorbic acid (also known asDL-ascorbic acid).

A skilled chemist will be able to select appropriate experimentalconditions in order to carry out the vinyl coupling reactions.

The swelling of the microgel particles can be initiated by varying thetemperature and/or pH. The temperature and/or pH required will depend onthe polymeric components of the microgel particles. Typically alltemperature responsive microgel particles will swell within atemperature range of 0 to 100° C., and suitably within the range of 20to 80° C. For in vivo applications where the in situ cross-linking ofthe microgel particles may be required, it is preferred that themicrogel particles swell at body temperature and/or the pH of the targettissue.

The vinyl coupling reaction may proceed at any suitable temperature.Temperatures ranging from 0 to 100° C., and suitably from 20 to 80° C.may be used. Again, for in vivo applications where the in situcross-linking of the microgel particles may be required, it is preferredthat the cross-linking reaction proceeds at normal body temperature.

The quantity of the microgel particles required for the vinyl couplingreaction is suitably 1 to 60 wt. %, and preferably from 10 to 20 wt. %,of the reaction medium.

The concentration of initiators should be in the range of 0.01 to 10 wt.% with respect to water. The preferred concentration is 0.1 to 2 wt. %with respect to water.

Any suitable pH range may be used for the vinyl-coupling reaction. ThepH range should include the pK_(a) for the microgel polymer if it is apH-responsive microgel particle. Again, for in vivo applications wherethe in situ cross-linking of the microgel particles may be required, itis preferred that the vinyl-coupling reaction proceeds at physiologicalpH. The pH range used during the binding of the microgel particles forpoly(MMA/MAA/EGDMA) microgel particles is 6.0 to 9.0, and is preferably7.0 to 8.0.

The swelling ratio (q=V/V_(coll)) defines the degree of swelling of themicrogel particles. V is the microgel particle volume measured in apartially swollen or fully swollen configuration. V_(coll) is the volumeof the non-swollen, collapsed configuration of the microgel particles.The value for q during vinyl-coupling reaction should be 1.1-500.Preferably, the value for q should be 3-100.

If ultraviolet photoinitiation is being used, then the intensity ofUV-irradiation required is, for example, half a minute to 2 hours ofexposure under a UV lamp providing a light intensity in the range of0.1-100 mW/cm². In a particular embodiment, the exposure is for 3minutes.

Cross-Linking of Microgel Particles by Forming an InterpenetratingPolymer Network

An alternative approach to bind the microgel particles together is shownschematically in FIG. 1. The first step of the reaction involvesproviding the microgel particles (without any vinyl-containing moietiesgrafted onto the particles). The next step involves bringing thesurfaces of the adjacent particles into contact with one another in thepresence of a cross-linking monomer comprising two or more vinyl groups.This can be achieved by causing the responsive microgel particles toswell (by varying the temperature or pH as described hereinbefore) inthe presence of the cross-linking monomer. The hydration and swelling ofthe microgel particles causes the surfaces of adjacent particles tocontact one another and overlap to form interpenetrating regions ofgelled polymer. The free-radical initiated polymerisation of thevinyl-containing cross-linking monomer can then be initiated. The resultis the formation of a cross-linked interpenetrating polymer networkwithin the swollen microgel particles. This network binds the microgelparticles together to form a cohesive gel structure.

By “interpenetrating polymer network” we mean that the polymer networkis formed within the swollen microgel particles and extends from onemicrogel particle to another. The polymer network is formed in situ andbetween the swollen particles by the polymerisation of the water solublemonomers that diffuse into the swollen microgel particles as theyhydrate.

An advantage of this method is that the addition of the water-solublecrosslinking monomer provides a useful tool for tuning the mechanicalproperties of the precursor. For instance, a low molar mass crosslinker(e.g., EGDMA) may be absorbed into the inter-penetrating microgels, andthereby links them together. The precursor microgel dispersion in thatcase is a physical gel.

A higher molar mass crosslinker (e.g., PEGDMA550) may be excluded fromthe microgel particle interior and, as such, an osmotic deswellingmechanism may be responsible for partially de-swelling the microgelparticles. In that case the precursor dispersion is a fluid (even whenthe pH has been increased). Crosslinking may result in formation of ahydrogel matrix that encapsulates the microgel particles. This networkmay to some extent inter-penetrate the peripheries of the microgelparticles.

It can be seen that the molar mass of the added water-soluble polymerplays a major role in the physical properties of the microgel dispersionprecursor and also the mechanical properties of the final DX microgels.

The vinyl polymerisation reaction can be carried out using thewater-soluble initiator as described above.

A water soluble accelerator as described herein may also be present.

Suitable reactions conditions for the polymerisation reaction will bewell known to those skilled in the art and reference is also made to thegeneral conditions described hereinbefore for the vinyl-couplingreactions.

Any suitable water-soluble cross-linking monomer may be used to form theinterpenetrating polymer network that binds the microgel particlestogether. For in vivo applications, it is necessary that the monomers(and the resultant interpenetrating polymer network formed) arebiocompatible. To be water-soluble a crosslinker may suitably have somewater solubility, for instance, ranging from 10⁻⁶ to 100 wt. % withrespect to the water phase. In particularly embodiments, the crosslinkerhas a water solubility of at least 0.1 wt %, suitably at least 1 wt %,or suitably at least 10 wt %.

Suitably, the cross-linking monomer will comprise two or more vinylgroups to enable a highly cross-linked interpenetrating polymer networkto be formed.

In an embodiment, the molar mass of the cross-linking monomer issuitably 220 to 750 g/mol, suitably 350 to 600 g/mol, or more suitably500 to 600 g/mol.

Suitably, the vinyl cross-linking monomer has the following formula:

wherein:

-   (a) R²¹, R²², R²³, R³¹, R³² and R³³ may be independently selected    from a group consisting of H; CH₃; a linear or branched alkyl group;    or a N-alkyl group of up to 10 C units; and wherein (b) R²⁴ may be    independently selected from a group consisting of:-   (i) —C(═O)—O—R³⁴—O—C(═O)—, wherein R³⁴ may comprise —CH₂—, —CH₂CH₂—    or a linear or branched alkyl group, such as a methylene chain,    which may be up to 20 C chains in length; or —C₆H₄—; or C₆H₃R³⁵,    wherein R³⁵ comprises substituents such alkyl, for example, CH₃; a    halogen group; or an amide group; or other di- or tri-substituted    phenyl groups containing more than one of these substitutents;-   (ii) —C(═)—O—R³⁶—C(═O)—, wherein R³⁶ may be —(CH₂CH₂O)— wherein n    may be from 1 to 30;-   (iii) —C(═O)—O—R³⁷R³⁸R³⁷—, wherein R³⁷ may comprise degradable ester    linkages, for example lactone, —[(CH₂)₅C(═O)—O]_(m)—, lactide,    —[CH(CH₃)C(═O)—O]_(m)—, glycolide, —[CH₂C(═O)—O]_(m)—, wherein m may    be from 1 to 50, and wherein R³⁸ may be —(CH₂CH₂O)_(n)—, wherein n    may be from 1 to 30;-   (iv) —C(═O)—O—R³⁹—, wherein R³⁹ may comprise degradable ester    linkages, for example lactone, [(CH₂)₅C(═O)—O]_(m)—, lactide,    [CH(CH₃)C(═O)—O]_(m)—, glycolide, [CH₂C(═O)—O]_(m)—, wherein m is    between 1 to 100;-   (v) allylacrylates, for example —C(═O)—O—R⁴⁰—, wherein R⁴⁰ may be    —CH₂—, —CH₂CH₂— or a linear, or branched, methylene chain up to 20 C    chains in length, or —C₆H₄—, C₆H₃R⁴¹, wherein R⁴¹ may comprise    substituents, such as alkyl, CH₃, a halogen or an amide group or    other di- or tri-substituted phenyl groups containing more than one    of these substitutents;-   (vi) vinylbenzenes, for example C₆H₄ or C₆H₃R⁴² wherein R⁴²    comprises substituents, such as alkyl; CH₃; a halogen or an amide    group (see (iii) above); or other substituted phenyl groups    containing more than one of these substitutents;-   (vii) acrylamides, for example C(═O)—NR⁴³—R⁴⁴—NR⁴⁵C(═O)—, wherein    R⁴³ and R⁴⁴ may be independently selected from a group consisting of    H; CH₃; a linear or branched alkyl group; a dialkyl group; a    N-alkylgroup, of up to 10 C units; and wherein R⁴⁴ may comprise    —CH₂—, —CH₂CH₂— or a linear, or branched, methylene chain up to 20C    chains in length; or —C₆H₄—, C₆H₃R⁴¹ wherein R⁴¹ comprises    substituents, such as alkyl; CH₃; a halogen or an amide group or    other di- or tri-substituted phenyl groups containing more than one    of these substitutents;-   (viii) trifunctional cross-linking monomers, wherein R²⁴ comprises    any of the groups listed in (b), as well as R²¹R²²C═CR²³, where R²¹,    R²² and R²³ are described in (a);-   (ix) tetrafunctional cross-linking monomers, wherein R²⁴ comprises    any of the groups listed in (b), as well as R²¹R²²C═CR²³ and    R³¹R³²C═CR³³, wherein R²¹, R²², R²³, R³¹, R³² and R³³ are described    in (a); and-   (x) wherein R²⁴ may contain any combination of the groups listed in    (b).

However, it is preferred that the monomer comprises a further functional(preferably, a di- or a higher functionality) cross-linking monomer suchas, for example, a substituted functional acrylate. Hence, the monomermay comprise allylmethacrylate or divinylbenzene. Hence, the monomer maycomprise butanediol diacrylate. However, preferably, the monomercomprises ethyleneglycol dimethacrylate.

The functional cross-linking co-monomer may have other groups in betweenthe terminal vinyl groups, for examplepoly(ethyleneglycol)dimethacrylate (PEGDMA).

The preferred vinyl cross-linking monomer from the point of view ofintervertebral disc repair is PEGDMA with a molar mass in the range of200 to 1000 g/mol. Preferably, the molar mass should be between 220 and750 g/mol, more preferably 300 and 600 g/mol. The most preferred vinylcross-linking monomer is EGDMA.

Cross-Linking of the Vinyl-Grafted Microgel Particles Combined With theFormation of an Inter-Penetrating Polymer Network

FIG. 3 shows a further alternative embodiment of the present inventionwhich is a combination of the approaches shown in FIGS. 1 and 2.

Thus, the compositions formed by this approach comprise a plurality ofmicrogel particles bound together by the reaction of vinyl-containingmoieties grafted onto the surfaces of the microgel particles and by across-linked polymer network that interpenetrates adjacent microgelparticles (and thereby further binds the particles together), whereinthe polymer network is formed by the polymerisation of a water solublecross-linking monomer comprising two or more vinyl groups.

In a particular embodiment, the microgel particles are formed frompoly(MMA/MAA/EGDMA) and glycidylmethacrylate is grafted onto the surfaceto provide the functional vinyl groups. These “functionalized” microgelparticles are then subject to a vinyl coupling reaction as definedherein in the presence of a cross-linking monomer, such as PEGDMA havinga molar mass in the range of 300 to 600 g/mol.

This method combines the benefits of directly cross-linking thevinyl-grafted microgel particles and cross-linking of microgel particlesby forming an interpenetrating polymer network. The interpenetratingnetwork may be considered a reinforcement of the directlydoubly-crosslinked microgel, but also provides a further means to tunethe mechanical properties of the precursor dispersion and DX microgelproperties using the molar mass of the water soluble crosslinker.

Properties of the Bound Microgel Particle Compositions of the Invention

The microgel compositions of the present invention belong to the classof materials known as hydrogels. They differ from conventional hydrogelsbecause they are composed of bound or linked microgel particles.

The elastic modulus (G) of the compositions of the invention will bedependent on the method used for their preparation. The values for G′,as measured by dynamic rheology, will typically be greater than 10 Pa(without any upper limit). The values for the loss modulus (G′) will beless than the elastic modulus for each composition of the inventionbecause of their classification as hydrogels.

The swelling characteristics of the compositions of the invention canagain be defined by the swelling ratio (as defined hereinbefore). Thevalue for q will typically be between 1.2 and 500. For the specificapplication of inter-vertebral disc repair, the swelling ratio ispreferred to be between 3 and 200.

The compositions of the invention, like the component microgelparticles, will be temperature and/or pH-responsive.Temperature-responsive compositions will be in the swollen configurationat temperatures below the LOST and in the collapsed configuration attemperatures above the LOST. The q value for such compositions willtypically be between 1.2 and 200. The elastic moduli will have the sameminimum values as specified above.

For pH-responsive compositions of the invention comprising microgelparticles composed of acidic monomers, the particles will be in aswollen configuration at pH values greater than the pK_(a) of the acidicmonomers and in the collapsed configuration at pH values less than thepK_(a) value of the acidic monomers. The pK_(a) values may be in therange of 1 to 13. The preferred range for the intervertebral discapplication is 5.0 to 8.0.

For pH-responsive compositions of the invention comprising microgelparticles composed of basic co-monomers, the particles will be in theswollen configuration at pH values less than the pK_(a) of the conjugateacid of the basic monomers and in the collapsed configuration at pHvalues greater than this pK_(a) value. The pK_(a) values may be in therange of 1 to 13.

The microgel compositions of the present invention suitably havesignificant critical strain values (γ*). The critical strain value isthe value for the strain, measured by a rheometer, at which the elasticmodulus (G) first reaches a value of 95% of that measured when γ=1.0%.The preferred range for γ* for the compositions of the invention is 2 to500%, more preferred is 5 to 300%, and even more preferred is 5 to 200%.

Applications

The compositions (including precursor compositions) of the presentinvention may be used for a variety of different applications, includingapplications in drug delivery, photonics, catalysis, informationstorage, or they may be used as absorbent materials.

It is envisaged that compositions (or precursor compositions) of thepresent invention will be particularly suitable for medicalapplications. It is particularly envisaged that compositions of thepresent invention may be used to repair damaged or degenerated softtissue in a subject.

Thus, the present invention provides a composition (or precursorcomposition) as defined herein for use in the treatment of damaged ordegenerated tissue, especially damaged or degenerated load-bearingtissues.

In a further aspect, the present invention provides a method of treatingdamaged or degenerated tissue, especially damaged or degeneratedload-bearing tissues, in a subject in need of such treatment, the methodcomprising administering a therapeutically effective amount of acomposition (or precursor composition) as defined herein.

Suitably, a composition of the invention is formed in situ within thebody (e.g. from a precursor composition). Therefore, the microgelparticles are administered together with any other materials required tobind the microgel particles together (e.g. reactants required for thevinyl coupling reactions) and thereby form a composition of the presentinvention in situ within the body. By “together with” we mean that oneor more of the reactants are either co-administered with the microgelparticles, administered before the microgel particles or administeredafter the microgel particles.

Thus, in one embodiment, the microgel particles having vinyl-containingmoieties grafted onto their surfaces are administered together with awater soluble initiator and, optionally, a water soluble accelerator.

In another embodiment, microgel particles (without vinyl-containingmoieties grafted onto the surface) are administered together with thecross-linking monomer (that forms an interpenetrating polymer networkfollowing polymerisation) and a water soluble initiator and, optionally,a water soluble accelerator.

In a further embodiment, microgel particles having vinyl-containingmoieties grafted onto their surfaces are administered together with avinyl cross-linking monomer (that forms an interpenetrating polymernetwork) and a water soluble initiator and, optionally, a water solubleaccelerator.

Additional components, such as a suitable vehicle, buffering agents,acids, bases, or other pharmaceutically acceptable excipients may alsobe administered together with the microgel particles and any otherreactants.

In one embodiment, the microgel particles are administered in thecollapsed or substantially collapsed configuration, which makes theparticle composition more fluid and therefore easier administer by, forexample, injection. When the microgel particles in the collapsedconfiguration reach the intended site, the particles preferably swell asa consequence of a change in the pH and/or temperature, and the vinylcoupling reaction can then be initiated to form a composition of thepresent invention in which the microgel particles are bound together.

In a particular embodiment, the microgel particles are pH-responsive andare adapted to be in a swollen or substantially swollen configuration atthe physiological pH of the target tissue. The pH of the administeredcomposition can then be manipulated so that the particles are in acollapsed configuration at the time of administration, but then swellwithin the target tissue. The rate of swelling may be increased by theadministration of a physiologically acceptable acid, base or buffersolution, either with or after the administration of the microgelparticles.

For example, if the microgel particles are being administered to an IVDin a subject, it is known that the average pH of the IVD is about 7.5.Microgel particles that are in a swollen or substantially swollenconfiguration at a pH of between 6.5 and 8, more preferably between 6.6and 7.5, would be particularly suitable for administration to an IVD.The pH of the microgel particle composition administered to the subjectcould then be manipulated so that the microgel particles are in thecollapsed or substantially collapsed configuration at the point ofadministration. In an embodiment, the microgel particles are maintainedat a pH of less than 6.6, such that upon administration into the IVD,the particles will swell and cause gelation of the composition. In suchan embodiment, the particles may be maintained in the collapsedconfiguration at a pH of between about 5.0 to 6.6, more preferablybetween about 5.5 to 6.6, and even more preferably between about 6.0 to6.6 before administration. At these pH values, the diameter of themicrogel particles is suitably between about 50-200nm, and mostpreferably, about 80 to 150 nm (as measured by Scanning ElectronMicroscopy). Accordingly, the increase in pH from less than 6.6 to about7.5 in vivo causes water to enter the particles such that they swell.When administered to a damaged or degenerated IVD, the swelling and insitu cross-linking of the microgel particles provides a composition ofthe invention which provides additional load bearing support to the IVD.

In an alternative embodiment, the microgel particles are administered ina swollen or substantially swollen configuration. Suitably, the microgelparticles are caused to swell just prior to administration. An advantageof this approach is that the microgel particles arrive at the targettissue in swollen form that is suitable for rapid cross-linking to forma composition of the invention. For example, pH-responsive microgelparticles may be administered in a swollen or substantially swollenconfiguration at a pH of, for example, 7.5 to 7.8.

In a particular embodiment, the microgel particles are administered inthe collapsed or substantially collapsed configuration, and allowed toswell in situ. The cross-linking reactants (the initiator and,optionally an accelerator and a cross-linking monomer (if aninterpenetrating polymer network is to be formed)) are added once themicrogel particles are sufficiently swollen for cross-linking to occur.

In a preferred embodiment, the microgel particles are administered inthe swollen or substantially swollen configuration and the cross-linkingreactants (the initiator and, optionally an accelerator and across-linking monomer (if an interpenetrating polymer network is to beformed)) are co-administered or administered immediately after theadministration of the microgel particles.

It may be preferred to contact the composition comprising the microgelparticle with a physiologically acceptable acid, base (e.g. NaOH or KOH)or buffer to facilitate a change in the pH and thereby acceleratinggelation in vivo. It will be appreciated that a physiologicallyacceptable acid, base or buffer may be administered to the target tissueeither before or after the composition comprising the microgel particlehas been administered. Alternatively, a co-administration procedure maybe used where both the composition comprising the microgel particles,and a physiologically acceptable buffer are administered substantiallyat the same time. This may be achieved for example through a speciallyconstructed syringe needle.

Suitably the reaction vinyl cross-linking reaction occurs promptly afterthe administration (before the initiator and optionally the acceleratordiffuse away from the site of administration). It is therefore preferredthat the initiator and optionally the accelerator are eitherco-administered with the microgel particles or administered immediatelyafter the microgel particles. Any cross-linking monomer that reacts toform an interpenetrating polymer network to bind the particles togethermay be co-administered with the microgel particles or administered priorto or after the administration of the microgel particles.

As a result of the in situ formation of the composition of the inventionwithin a subject, there is preferably, an increase in disc height andalso the Young's Modulus of the IVD as the composition forms, and themechanical strength is effectively restored. Advantageously, thisprovides a minimally invasive method that can fill the interior of anyirregularly shaped clefts in the IVD. Hence, this minimally invasivemethod does not involve any major surgical intervention, thereby meaningthe subject being treated is likely to have a much curtailed recoverytime.

Another advantage of the method is that it does not require removal ofany healthy tissue. This is in direct contrast to nucleus replacementtechnologies which involve microdiscectomy and removal of nucleuspulposus tissue.

In another embodiment, the composition administered to form thecomposition of the invention may comprise at least one nucleus pulposuscell and/or at least one stem cell and/or at least one mammalian cell.

Examples of suitable mammalian cells, which may be added to thecomposition include chondrocytes (e.g. autologous or autogenous).Examples of suitable stem cells, which may be added to the compositioninclude mesenchymal, haematopoeic etc., including embryonic and clonedstem cells. In addition, the composition administered may furthercomprise collagen and/or proteoglycans. It will be expected that addingnucleus pulposus cells to the composition will increase the rate ofrecovery of the subject. Hence, a further advantage of the methodaccording to the invention is that it allows mixing of living cells(e.g, NP cells or stem cells) with the composition comprising themicrogel particle dispersion in order to facilitate re-growth of NPtissue. Thus, the method according to the invention is amenable tocombining mechanical support with a biological repair system.

Examples of suitable soft tissues which may be treated include skin,muscle, ligament, or adipose tissue. Such damaged or degenerated softtissue may comprise a wound, which may be either acute or chronic.However, it is preferred that the soft tissue being treated comprisesdamaged or degenerated load-bearing tissue such as, for example,intervertebral discs and the tissues found in articular joints (such asthe elbow, knee, hip, wrist, shoulder and ankle). In addition, thecompositions of the invention may be used to treat low-load bearingjoints, such as, for example, the joints present in a finger or a thumb.

It is most preferred that the compositions of the present invention areused to treat damaged or degenerated vertebral, or intervertebral discs(IVDs). Preferably, the method of the second aspect comprisesadministering the composition directly into the IVD, and preferably intothe nucleus pulposus (NP) thereof. Hence, advantageously, no surgery isrequired using this approach. More preferably, the composition may beadministered directly into clefts within the NP, which form when theproteoglycan content in the IVD decreases with age. Furthermore, it ispreferred that the components required to form the composition of theinvention are administered by injection into the target tissue.

Disease conditions, which may be treated with the medicament of thefirst aspect or the method according to the second aspect includearthritis, intervertebral disc degeneration, back pain, low back pain,sciatica, cervical, spondylosis, neck pain, kyphosis, scoliosis,degenerative joint disease, osteoarthritis, spondylolysis,spondylolisthesis, prolapsed intervertebral disc, failed spine surgery,and spinal instability. The disease condition may be chronic or acute,for example, chronic or acute back pain.

In a particular embodiment, the composition of the invention is used forthe treatment of osteoarthritic conditions in joints as an alternativeto cartilage replacement. In such embodiments, the composition of theinvention is formed by in situ within the joint by injecting thecomponents required to form the composition of the invention into thejoint capsule of an osteoarthritic joint. The composition of theinvention would then provide a means of keeping the bone ends apart.

The swelling pressure of the gels of the present invention can beadjusted using pH and/or temperature in order to increase the effectiveYoung's Modulus of the microgel-loaded soft tissue. Furthermore, theinventors believe it should be possible to adjust the pKa of thesemicrogels by varying the chemical composition of the particles, whichwill allow fine-tuning of the load-bearing properties of these materialsat the pH of the damaged load-bearing tissue.

It will be appreciated that the composition according to the presentinvention may be used in a monotherapy (i.e. use of the compositionaccording to the invention alone to prevent and/or treat diseasescharacterised by damaged or degenerated soft tissue, and preferably,load-bearing tissue). Alternatively, the microgel particle according tothe invention may be used as an adjunct, or in combination with otherknown therapies.

Compositions comprising the microgel particle according to the inventionmay be used in a number of ways. Preferably, the composition may beadministered by injection.

The therapy may be given as a single administration (e.g. a singleinjection). Alternatively, the composition used may require repeatedadministration at predetermined intervals.

The invention further provides a pharmaceutical composition comprising atherapeutically effective amount of a microgel particle together withreactants to form the composition of the invention in situ within thebody. A “therapeutically effective amount” is any amount of a microgelparticle according to the invention which, when administered to asubject forms a composition of the invention that prevents and/or treatsa disease characterised by damaged or degenerated soft tissue.

In a further aspect, the present invention provides a kit of partscomprising microgel particles as defined herein (optionally in thepresence of a suitable vehicle/dispersion medium) and one or morecross-linking reactants as defined herein. The reactants suitablecomprise an initiator as hereinbefore defined, and may further comprisean accelerator and/or a cross-linking monomer as hereinbefore defined.Suitably, the kit further comprises instructions explaining how toadministered the contents for in situ gel formation.

Throughout the description and claims of this specification, the words“comprise” and “contain” and variations of them mean “including but notlimited to”, and they are not intended to (and do not) exclude othermoieties, additives, components, integers or steps. Throughout thedescription and claims of this specification, the singular encompassesthe plural unless the context otherwise requires. In particular, wherethe indefinite article is used, the specification is to be understood ascontemplating plurality as well as singularity, unless the contextrequires otherwise.

EXAMPLES

The invention will now be described in more detail in relation to thefollowing illustrative examples.

Physical Measurements

Unless stated otherwise, the following methodology was used to obtainphysical measurements.

Titration measurements were performed using a Mettler titration unit inthe presence of a supporting electrolyte (0.1 M NaCl). Photoncorrelation spectroscopy measurements were performed using dispersionscontaining φ_(p)=3×10⁻⁴ microgel. The measurements were conducted usinga BI-9000 Brookhaven light scattering apparatus (Brookhaven InstrumentCooperation), fitted with a 20 mW HeNe and the detector was set at 90°scattering angle. The extent of particle swelling is characterised interms of the estimated swelling ratio, Q. This is given by the followingequation.

$\begin{matrix}{Q = ( \frac{d}{d_{coll}} )^{3}} & (1)\end{matrix}$

For equation (1) d and d_(coll) are the diameters of the measured usingphoton correlation spectroscopy (PCS) at a given pH and the collapsedparticle size, respectively. In this work the values for d_(coll) werethose obtained at pH=4 unless otherwise stated. SEM measurements wereobtained using a Philips FEGSEM instrument. Samples were dried at roomtemperature or by freeze drying. At least 100 particles were counted forparticle size estimations. Dynamic rheology measurements were performedusing a TA instrument AR G2 temperature-controlled rheometer with anenvironmental chamber. A 20 mm diameter plate geometry with a solventtrap was used. The gap was 1000 nm.

Swelling experiments for the DX (cross-linked) microgels of the presentinvention were performed by placing samples in buffer and then allowingthe sample to equilibrate with gentle agitation for a period of at 8days. The buffer was regularly changed. Periodically, the sample wasremoved, patted dry with paper towel, weighed and then immediatelyreturned to the buffer solution. The buffer solutions used werephosphate or phthalate based, had an ionic strength of about 0.1 M, andwere prepared as described elsewhere (J. Brandrup, E. H. Immergut, E. A.Grulke, A. Abe, and D. R. Bloch (1999) CRC Polymer Handbook, 4 ed., JohnWiley & Sons). The volume swelling ratio for those DX microgels (Q_(DX))were determined gravimetrically and calculated using:

$\begin{matrix}{Q_{DX} = {{\rho_{p}( {\frac{Q_{{DX}{(m)}}}{\rho_{s}} + \frac{1}{\rho_{p}}} )} - \frac{\rho_{p}}{\rho_{s}}}} & (2)\end{matrix}$

For equation (2) Q_(DX(m)) is the ratio of the swollen gel mass to thedry mass. ρ_(s) and ρ_(p) are the densities of the solvent and polymer,respectively. These were taken as 1.2 and 1.0 gcm⁻³.

Method 1—Preparation of Poly(MMA/MAA/EGDMA) Microgel Particles

Poly(MMA/MAA/EGDMA) was prepared using emulsion polymerisation. A waterbath was heated to 80° C. 1.8 g sodium dodecylsulfate (SDS) wasdissolved in 517.5 g deionised (DI) water. The solution was thenfiltrated to a four necked flask and nitrogen purged for 30 minutes. Themonomer mixture was prepared with following composition: MMA (189.76 g),MAA (94.87 g) and EGDMA (2.882 g). This includes 15% excess in order toaccount for loss during the feed stage. After SDS dissolution, 31.5 g ofmonomer mixture (seed) was added, whilst stirring, to the vesselfollowed immediately by adding respectively K₂PO₄ solution (0.2264 g in2.93 g DI water) and ammonium persulfate (APS) solution (0.2 g in 3.39 gDI water). The mixture was left to stir for a further 30 minutes andtemperature was raised to 88° C. The remaining monomer mixture was addedover 90 min at a rate of approximately 2.5 ml/min. After the feed wascomplete an APS solution was added (0.0874 g in 3 g DI water). Thedispersion was left to stir at least for 2 hours until no monomers couldbe detected and then cooled with water and ice mixture while beingstirred. The cooled product (a milky dispersion) was filtered. Thismicrogel contained a nominal concentration of 66 wt. % MMA, 33 wt. % MAAand 1 wt. % EGDMA.

Method 1A—Preparation of Poly(MMA/MAA/EGDMA) Microgel Particles

Poly(MMA/MAA/EGDMA) was also prepared using the following method. 1.8 gSDS in 517.5 g DI water was added to a four-necked round bottom equippedwith a mechanical stirrer and reflux condenser. The contents were purgedwith nitrogen for 30 minutes at 80° C. To form the seed, 31.5 g of asolution of MMA (66 wt %), MAA (33 wt %) and EGD (1 wt %) was added tothe vessel followed immediately by adding, respectively, K₂HPO₄ (3.15 gof 7 wt. % solution) and ammonium persulfate (APS, 3.5 g of 5 wt. %solution). The seed was left to stir for a further 30 minutes and thetemperature was raised to 85° C. The remaining 218.5 g of monomermixture (with same proportions as above) was added uniformly over 90minutes with rate of approximately 2.43 g/min. After the additionanother portion of APS (3.1 g of 3 wt. % solution) was added and thereaction was continued for another 2 hours. The product was cooled incold water with stirring. After filtration, the microgels were dialysedin DI water for 14 days (DI water was changed twice a day).

Method 2—Preparation of Poly(EA/MAA/BDDA) Microgel Particles

Poly(EA/MAA/BDDA) microgel was prepared using the seed-feed (starvedfeed) emulsion polymerisation method. A monomer mixture containing EA(Aldrich, 99%, 143.5 g), MAA (Aldrich, 99%, 72.0 g) and BDDA (Aldrich,98%, 2.2 g) was prepared and 12.5% of the mixture added to a pre-purged,stirred, solution of sodium dodecylsulfate (BDH, 1.75 g in 500 g ofwater) which had been heated to 80° C. The monomers were passed over analumina column prior to use to remove the inhibitor. K₂HPO₄ (3 g of 7%solution in water) and 2.95 g of a 5% ammonium persulfate solution inwater were immediately added whilst maintaining a nitrogen atmosphere.After appearance of a slight blue turbidity, the remaining monomermixture was added at a continuous rate over a 90 min period. Additionalammonium persulfate (3.3 g of 5% solution in water) was added and thetemperature maintained at 80° C. for a further 2 h. The microgel wasextensively dialysed against Milli-Q quality water.

Method 2A—Preparation of Poly(EA/MAA/BDDA) Microgel Particles

The poly(MMA/MAA/BDDA) microgel was also prepared using a similar methodto Method 1A. However, MMA and EGD were replaced by EA and BDD,respectively, at the same mol. %.

Method 3—GMA Functionalisation of Poly(MMA/MAA/EGDMA) Microgel

Before glycidyl methacrylate (GMA, purum, ≧97.0% (GC))functionalisation, poly(MMA/MAA/EGDMA) microgel [Method 1] was purifiedextensively by dialysis with changing the deionised water twice a dayfor at least two weeks. The purified microgel dispersions was mixed withGMA. This was done at a concentration of 5 times of GMA to thecarboxylate concentration present in the microgel. The mixtures werediluted with DI water to microgel concentration 14.87 wt %, and the pHvalue was adjusted to 3.5 by adding aqueous HCl solution. The system wasreacted at 50° C. in a water bath by stirring for 8 h. The reactionmixture was washed by ethyl acetate 4 times to remove most of unreactedGMA, and further dialyzed 3 days to remove any unreacted GMA completely.

Method 3A—GMA Functionalisation of Poly(MMA/MAA/EGDMA) andPoly(EA/MAA/BDDA) Microgel

GM-M-EGD and GM-E-BDD refer to the GM-functionalised M-EGD and E-BDDmicrogels, respectively. The method used for functionalisation of M-EGDis briefly described in the following. The pH of a M-EGD dispersion witha polymer volume fraction (□p) of 0.15 containing 1.73 M of GM wasadjusted to 3.5, and then heated to 50° C. with mechanical stirring for8 h. The dispersion was washed four times with ethyl acetate anddialysed extensively to obtain the purified GM-M-EGD. A similarprocedure was used to prepare GM-E-BDD particles. However, in that casethe pH was 5.1 during functionalisation and the particles were washedwith chloroform prior to dialysis. A pH of 5.1 was also used for thepreparation of a more highly functionalised GM-M-EGD microgel, which isabbreviated here as GM(H)-M-EGD.

Method 3B—AEMHCL Functionalisation of Poly(MMA/MAA/EGDMA) andPoly(EA/MAA/BDDA) Microgel

A typical preparation is described as follows for the preparation ofAEM-functionalised M-EGD with a [AEM]/[MAA] ratio of 0.40, i.e.,AEM40-M-EGD. The concentration ratio of MAA:EDC:NHS:AEM was1:0.5:0.4:0.4. EDC and NHS areN-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride andN-hydroxysuccinimide, respectively. 10.25 g of 29 wt % M-EGD was placedin a 100 ml round bottom flask with magnetic stirrer and diluted by 30ml of pH =6.6 buffer (0.1 M). Then 0.53 g of NHS was dissolved in 5 mlof buffer, 0.88 g of EDC was dissolved in 5.25 ml of 1M HCl. Thesolution containing NHS and EDC was then added to the microgeldispersion and allowed to stir for 20 min. Then, 0.76 g of aminoethylmethacrylate hydrochloride (AEMHCI) was dissolved in 5 ml of buffer andadded. The final pH was adjusted to 6.5 by further buffer addition. Thereaction was allowed to proceed for 1 day at RT. The product waspurified by repeated centrifugation and re-dispersion in Milli-Q gradewater. The partially aggregated state of the microgels facilitatedcentrifugation using a conventional high speed centrifuge.

AEM-E-BDD functionalised microgels were prepared in substantially thesame manne, except that the pH was adjusted to about 7.0.

Method 4—Calculation of the mol. % of GMA Grafted on toPoly(MMA/MAA/EGDMA) Microgel Particles in Method 3

These data were obtained by titration of the free carboxylic acid groupson the microgel particles and calculation of the mol. % of those groupsreacted. Comparison with the composition of the microgel (Microgel 2B)enabled calculation of the mol. % of GMA incorporated. The results areshown in Table 1 below (see Method 6).

Method 4A—Calculation of the mol. % of GMA Grafted on toPoly(MMA/MAA/EGDMA) and Poly(EA/MAA/BDDA) Microgel Particles in Method3A

As in Method 4 above, these data were obtained by potentiometrictitration. The mol % GMA was determined from the difference in the mol %MAA in the microgel before and after functionalisation. The results areshown in Table A below.

TABLE A Mol. % Mol. % Code MAA^(a) GMA^(a) M-EGD 35.9 — E-BDD 37.2 —GM-M-EGD 34.1 1.8 GM(H)-M-EGD 35.9 5.8 GM-E-BDD 26.5 7.8 ^(a)Determinedfrom potentiometric titration data. The mol. % GMA was determined fromthe difference in the mol. % MAA in the microgel before and afterfunctionalisation.

Method 4B—Calculation of the mol. % of AEMHCI Grafted on toPoly(MMA/MAA/EGDMA) and Poly(EA/MAA/BDDA) Microgel Particles in Method3B

As per Method 4, these data were obtained by potentiometric titration.The mol % AEMHCL was determined from the difference in the mol % MAA inthe microgel before and after functionalisation. The results are shownin Table A below.

TABLE B Mol. % Mol. % [AEM]/ MAA AEM Code [MAA]^(a) (exp)^(b) (exp)^(c)M-EGD — 42.5 — AEM5-M-EGD^(g) 0.05 38.7 3.0 AEM10-M-EGD 0.10 38.5 4.1AEM20-M-EGD^(g) 0.20 34.3 7.4 AEM30-M-EGD 0.30 31.1 11.4 AEM40-M-EGD0.40 35.1 7.4 AEM50-M-EGD 0.50 31.5 11.0 E-BDD — 37.2 — AEM20-E-BDD 0.20^(a)Concentration ratio of AEMHCI and MAA used to prepare thefunctionalised microgels. ^(b)Mol. % MAA found in the microgels bytitration. ^(c)AEM contents determined from pH titration data for themicrogels using the difference between the MAA contents in the parentmicrogel and the respective AEM-functionalised microgel.

Potentiometric titration was used to determine the MAA content and theeffective pK_(a) values for all of the microgels studied. See Table Babove. These data also enabled calculation of the mol. % of AEM withinthe functionalised microgels.

FIG. 5B-1 shows variation of Mol. AEM within the AEM-M-EGD microgelswith [AEM]/[MAA]. The broken line represents the theoretical values for100% efficiency of functionalisation.

It can be seen from FIG. 5B-1 that very good agreement between theoryand experiment was observed until [AEM]/[MAA] exceeded 0.30. At higher[AEM]/[MAA] levels a much lower than expected incorporation occurred andthe values became more variable. Interestingly, the maximum mol. % ofAEM incorporated did not exceed the value determined at the end of thelinear region (11.4 mol. %).

FTIR data were obtained for the functionalised microgels (FIG. 5B-2).

FIG. 5B-2 shows selected FTIR spectra for AEM-M-EGD microgels. Thelegend shows the [AEM]/[MAA] ratios used. The spectra were recorded ondry films and selected bands are labelled.

They show evidence of vinyl group incorporation from a band at 1020cm⁻¹, which is present for the AEM-functionalised microgels but absentin the spectrum for M-EGD. The Amide I (1647 cm⁻¹) and Amide II (1560cm⁻¹) bands¹⁷ were also evident upon functionalisation. The spectrasupport our interpretation that vinyl functionalisation occurredsuccessfully.

The pK_(a) values for the microgels were calculated at eachneutralisation point from the titration data using:

${pK}_{a} = {{pH} - {\log ( \frac{\propto}{{1 -} \propto} )}}$

where α is the degree of neutralisation. We (and others) haveestablished that as the MAA content in latex particles increases thepK_(a) decreases. This has been attributed (Pinprayoon, O.; Groves, R.;Saunders, B. R. J. Coll. Interf. Sci. 2008, 321, 315) to a decrease inhydrophobic interactions which oppose particle swelling as a result ofionisation.

FIG. 5B-3 shows variation of pK_(a) with neutralisation for AEM-M-EGDmicrogels. The legends give the [AEM]/[MAA] ratios used for theirpreparation. The data show a pronounced increase in the pK_(a) valuesfor α<20% with increasing [AEM]/[MAA]. This is a strong indication thatfunctionalisation proceeds from the exterior of the microgel inwards.The AEM groups are relatively hydrophobic compared to MAA and this leadsto an increase of the local pK_(a). These data provide evidence of alocally high AEM functionalisation at the microgel periphery. This isconsistent with the particles being partially swollen at the initialstages of the functionalisation process. It is likely that theperipheries of the particles close up as the functionalisation proceedsdue to an increased hydrophobicity and loss of charge in that region.This would cut off supply of AEM to the inner regions of the microgels.

Method 5—pH-Dependent Particle Size Measurements for Microgels fromMethods 1 and 2

The measurements were performed using photon correlation spectroscopyusing a particle concentration in the range of 0.001 to 0.1 wt. %Standard buffer solutions were used for these experiments. Thesemeasurements were performed using a Brookhaven BI-9000 light scatteringapparatus fitted with a 20 mW HeNe laser. The detector was set at a 90°scattering angle.

The results are shown in FIG. 4 [Microgel 1 (open diamonds), 2A (opentriangles), 2B (open squares), 2BG (closed squares)].

Method 5A —pH-Dependent Particle Size Measurements for Microgels fromMethods 1A, 2A and 3A

The measurements were performed using photon correlation spectroscopyusing dispersions containing φ_(p)=3×10⁻⁴ microgel. Standard buffersolutions were used for these experiments. These measurements wereperformed using a Brookhaven BI-9000 light scattering apparatus fittedwith a 20 mW HeNe laser. The detector was set at a 90° scattering angle.

FIG. 4A shows the pH-dependence of the hydrodynamic diameter (d_(h)) andQ values for the microgels: a) and b) [M-EGD (solid diamonds), GM-M-ECD(open squares)]; c) and d) [E-BDD (solid diamonds), GM-E-BDD (opensquares)], where GM-prefix refers to a GMA functionalized microgel.

Selected data are also shown in Table 1. The data show pH-triggeredswelling at pH of about 6.4. Complete swelling had occurred by pH ofabout 7.4. The latter corresponds to the pK_(a) of the microgel. It isnoted that there is not perfect agreement between the pK_(a) values andthe particle swelling data (FIG. 4A). The pK_(a) for polyelectrolytegels is strongly affected by a number of factors, which includes polymerand electrolyte concentration¹⁸. Therefore, the differences between thepK_(a) values and the pH range of strong swelling (FIG. 4A) can beattributed to differences in electrolyte concentration and polymerconcentration within the microgel particles for each technique.

Interestingly, it can be seen from FIG. 4A that the GM-M-EGD microgelsswell much more than the M-EGD particles. This was suspected to be dueto the ethyl acetate washing procedure, which was used to removeresidual GMA from the microgels. To test this idea non-functionalizedmicrogel was washed with ethyl acetate. The particle size for M-EGDincreased from to 285 nm at pH=8 after ethyl acetate washing (cf. 232 nmin Table 1). This is a new observation for pH-responsive MMA-basedmicrogels and strongly indicates that reversible hydrophobic associationrestricts the swelling for these microgels. The washing process mustremove physical crosslinks, presumably involving MMA groups. Thepresence of hydrophobic crosslinks could be aided by the low mobility ofPMMA chains due to their high T_(g). Ethyl acetate may act as aplasticiser for the M-EGD particles. In contrast the pH-dependentswelling for the E-BDD and GM-E-BDD microgels were almost identical(FIG. 4A(b) and 4A(d). This is probably because the E-BDD microgels iscomposed of polymer chains with a lower T_(g) and is less affected byhydrophobic physical crosslinks. This is supported by the higher Qvalues obtained for the E-BDD microgels (FIG. 4A).

FIG. 4A-2 includes additional data showing the variation of (a)hydrodynamic diameter and (b) swelling ratio with pH for the variousmicrogels, including the highly functionalized GM(H)-M-EGD microgel.

Method 5B—pH-dependency and other depencies of particle sizemeasurements for microgels from Methods 1A, 2A and 3B

The measurements were performed using photon correlation spectroscopyusing dispersions containing φ_(p)=3×10⁻⁴ microgel. Standard buffersolutions were used for these experiments. These measurements wereperformed using a Brookhaven BI-9000 light scattering apparatus fittedwith a 20 mW HeNe laser. The detector was set at a 90° scattering angle.

FIG. 4B-1 shows variation of (a) hydrodynamic diameter, (b) swellingratio and (c) φ_(p)* with pH for the M-EGD (solid diamonds) and E-BDD(open triangles) microgels. The data were measured using 0.1 M buffers.

The microgel particles swell when the pH approaches their respectivepK_(a) values. Using equation (1) the maximum values of Q for M-EGD andE-BDD are 4 and 40, respectively. The E-BDD microgel swell verystrongly. The relatively low swelling for the M-EGD microgels is becausethey have a reversible crosslinking contribution which restrictsswelling (Liu, R.; Milani, A. H.; Freemont, T. J.; Saunders, B. R.Manuscript submitted to Soft Matter 2011). The swelling can be increasedgreatly for the M-EGD microgels using ethylacetate washing.

The polymer volume fraction at which neighbouring microgel particlesoverlap, φ_(p)*, because gives an indication of when inter-particlecrosslinking may be effective. Also, it will be related to the point atwhich physical gelation occurs. In recent work (Lally, S.; Cellesi, F.;Freemont, T.; Saunders, B. R. Coll. Polym. Sci. 2011, In Press) we foundthat φ_(p)* could be estimated for E-BDD microgel dispersions as thevalue of φ_(p) which is equal to the internal polymer volume fractionwithin the (swollen) microgel particles (φ_(MG)). Our approach formicrogels draws upon the definition of polymer overlap concentrationsfor polymer micelles (van Ruymbeke, E.; Pamvouxoglou, A.; Vlassopoulos,D.; Petekidis, G.; Mountrichas, G.; Pispas, S. Soft Matter 2010, 6,881). It assumes that the radial segment distribution within eachparticle is constant and ignores interstitial voids between theparticles, i.e. assumes a packing efficiency of 100%. For hard spheremicrogel particles a packing efficiency of less than 100% is expected³.However, the precise packing efficiency will be dependent on microgelcomposition and is not able to be pre-determined. The following equationwas used to estimate φ_(p)*.

$\begin{matrix}{\varphi_{p}^{*} = \frac{1}{Q}} & (3)\end{matrix}$

Data for φ_(p)* are shown in FIG. 4B-1( c). These data can be used toinfer overlap and also gel elasticity. It is expected that overlap andelasticity will be highest for the E-BDD physical gels if φ_(p) value of0.10 is used and the pH is greater than 6.5.

FIG. 4B-2 shows variation of (a) hydrodynamic diameter with pH, (b)diameter with [AEM]/[MAA] ratio and (c) nominal Q with [AEM]/[MAA] forthe AEM-M-EGD microgels. The data point in (b) for [AEM]/[MAA]=0.30 andlabelled as pH=8 was measured at pH=7.4. The data in (a) are forAEMHCL-M-EGD microgels formed using an [AEM]/[MAA] concentration ratioof 0.5 (open triangles), 0.1 (solid diamonds), 0 (open diamonds)], wherean AEMHCL-prefix refers to an AEMHCL functionalized microgel.

PCS was used to measure the size of the collapsed particles (pH=4) afterfunctionalisation. The data are shown in FIG. 4B-2( a) and (b) Swellingratio data are shown in FIG. 4B-2( c), where the term Q_(nom) has beenused for Q.

The data show a linear increase in diameter with [AEM]/[MAA] ratio forall of the M-EGD microgels studied at pH=4 and 8. This is attributed toaggregation. Nominal Q values at pH=8 were calculated using equation (1)and are shown in FIG. 4B-1( c). These values, which are subject toconsiderable scatter, are based on the assumption that the aggregatesare irreversibly formed during the vinyl functionalisation stage and donot break down when the pH is increased. The fact that Q_(nom) does notchange significantly with [AEM]/[MAA] indicates that the swelling of theindividual particles has not been greatly affected by AEMfunctionalisation. That is they have maintained their pH-responsiveness,even in the aggregated state. See also FIG. 4B-1( a). This is reasonablegiven that the maximum extent of functionalisation affects about 1/3 rdof the MAA groups present in the parent microgel (Table 1).

In contrast to an earlier vinyl-functionalisation method (Example 3A)involving GMA (glycidyl methacrylate) the method used here involved ahigh electrolyte concentration. This is because of the use of AEMHCI.The pH used in this method was ca. 6.6, which required addition ofbuffer and NaOH. This pH was used to ensure the microgel particles wereat least partially swollen during functionalisation. AEMHCI is anelectrolyte and so high ionic strengths were present.

FIG. 4B-3 shows variation of (a) hydrodynamic diameter and (b) swellingratio (Q) with concentration of NaCl in solution at pH=6.6 for M-EGDmicrogel dispersion.

The effect of added NaCl on the particle size of the microgel wasinvestigated at pH=6.6 (FIG. 4B-3). At this pH the particles wereslightly swollen (Q=1.5). A slight increase in size occurred (to ca. 170nm, FIG. 3S) at a NaCl concentration of 0.10 M indicating some slightaggregation. These data show that functionalisation occurred underconditions where the particles were (initially) slightly swollen andthat limited (partial) aggregation occurred. The extent of aggregationwill depend on the extent of particle swelling (and hence pH). Forcomparison, functionalisation using [AEM]/[MAA]=0.40 involved additionof reactants and buffer that gave an ionic strength contribution of 0.28M. An additional contribution from the microgel particles (MAA groups)themselves would have increased the ionic strength. Therefore, it is notsurprising that partial aggregation occurred during thefunctionalisation process. The data from FIG. 4B-2 show this becameprogressively more pronounced with increasing [AEM]/[MAA]. It isimportant to note that the aggregate sizes from FIG. 4B-2 are all lessthan 1 μm. This means that these dispersions would still able to beinjected through narrow gauge syringe needles. This is important forfuture potential applications involving minimally invasive techniques(e.g., injection).

Method 6—Characterisation of the Microgel Particles Prepared in Methods1, 1A, 2, 2A, 2B, 3, 3A, and 3B

The characterisation data is presented in Table 1 below:

TABLE 1 d_(h(4))/ d_(h(10))/ No. Method Composition ^(a) nm ^(b) nm ^(b)q ^(c) pK_(a) 1 2 poly(EA₆₆/MAA₃₃/BDDA_(1.0))  75 309   70   6.70 2A 1poly(MMA₆₆/MAA₃₃/EGDMA_(1.0)) ^(d) 104 205    7.7 2B 1poly(MMA₆₆/MAA₃₃/EGDMA_(1.0)) 130 208    4.1 6.35 2BG 3poly(MMA₆₆/MAA₂₃/EGDMA_(1.0))-GMA_(0.018) 131 323 ^(g) 15 ^(h) 7.1 3^(e) 1 poly(MMA₆₆/MAA₃₃/PEGDMA550_(1.0)) 150 3G ^(e) 3poly(MMA₆₆/MAA₃₃/PEGDMA550_(1.0))- GMA ^(f) M-EGD 1Apoly(MMA₆₆/MAA₃₃/EGDMA_(1.0)) 139 232 ^(g)   4.7 ^(h) 7.4 E-BDD 2Apoly(EA₆₆/MAA₃₃/BDDA_(1.0))  75 247 ^(g) 35 ^(h) 6.5 GM-M- 3Apoly(MMA₆₆/MAA₃₃/EGDMA_(1.0))-GMA_(0.018) 131 323 ^(g) 15 ^(h) 7.1 EGDGM(H)- 1A poly(MMA₆₆/MAA₃₃/EGDMA_(1.0))-GMA_(0.058) 133 315   13 6.0M-EGD GM-E- 3A poly(EA₆₆/MAA₃₃/BDDA_(1.0))-GMA_(0.078)  77 243 ^(g) 32^(h) 6.1 BDD M-EGD 1A poly(MMA₆₆/MAA₃₃/EGDMA_(1.0)) 134 212 ^(g) 7.2AEM5- 3B poly(MMA₆₆/MAA₃₃/EGDMA_(1.0))-AEM_(0.030) — — 6.5 M-EGD AEM10-3B poly(MMA₆₆/MAA₃₃/EGDMA_(1.0))-AEM_(0.041) 257 306 ^(g) 6.6 M-EGDAEM20- 3B poly(MMA₆₆/MAA₃₃/EGDMA_(1.0))-AEM_(0.074) — — 6.7 M-EGD AEM30-3B poly(MMA₆₆/MAA₃₃/EGDMA_(1.0))-AEM_(0.114) 653 914 ^(g) 6.8 M-EGDAEM40- 3B poly(MMA₆₆/MAA₃₃/EGDMA_(1.0))-AEM_(0.074) 655 742 ^(g) 6.7M-EGD AEM50- 3B poly(MMA₆₆/MAA₃₃/EGDMA_(1.0))-AEM_(0.11) 576 851 ^(g)6.9 M-EGD AEM20- 3B poly(MMA₆₆/MAA₃₃/EGDMA_(1.0))-AEM E-BDD μ-BDD 2poly(EA₆₆/MAA₃₃/BDDA_(1.0)) 108 310 ^(i) 24 ^(j)  6.7 ^(a) The numbersin the subscripts are the approximate nominal compositions (wt. %) basedon the preparation conditions. In the case of GMA or AEM the number isbased on titration data and refers to the composition as a whole (SeeExample 10). ^(b) Hydrodynamic diameter measured at pH = 4 or 10. ^(c)Volumetric swelling ratio calculated using d_(h(10)) and d_(h(4)) valuesaccording to: q = (d_(h(10))/d_(h(4)))³. ^(d) This microgel was preparedusing high shear. ^(e) 3 and 3G were made using methods 1 and 3,respectively, The difference is that PEGDMA550 was used instead ofEGDMA. The wt. % concentration was the same. ^(f) The composition of 3Gshould be similar to that of Microgel 2BG, but has not been establishedat the present time. ^(g) In these cases, the hydrodynamic diameter wasmeasured at pH 8, not 10. ^(h) In these cases, volumetric swellingratios are calculated using d_(h(8)) and d_(h(4)) values according to: q(d_(h(8))/d_(h(4)))³. ^(i) In these cases, the hydrodynamic diameter wasmeasured at pH 7, not 10. ^(j) In these cases, volumetric swellingratios are calculated using d_(h(7)) and d_(h(4)) values according to: q= (d_(h(7))/d_(h(4)))³. The extent of functionalisation by GMA forGM-M-EGD (from method 3A) was modest (1.8 mol. % overall, or 5% of theMAA groups). As will be shown below this was sufficient to form DX gels.In the case of the GM-E-BDD (from method 3A) the functionalisation wasmuch higher (7.8 mol. % overall, or 21% of the MAA groups). To preparecolloidally stable GM-E-BDD microgel dispersions it was necessary to usea higher pH for the functionalisation to avoid aggregation during theprocess. The relatively high GMA content for the GM-E-BDD microgels didnot decrease the extent of swelling as judged by the Q values at pH = 8(Table 1).

^(j)In these cases, volumetric swelling ratios are calculated usingd_(h(7)) and d_(h(4)) values according to: q=(d_(h(7))/d_(h(4)))³. Theextent of functionalisation by GMA for GM-M-EGD (from method 3A) wasmodest (1.8 mol. % overall, or 5% of the MAA groups). As will be shownbelow this was sufficient to form DX gels. In the case of the GM-E-BDD(from method 3A) the functionalisation was much higher (7.8 mol. %overall, or 21% of the MAA groups). To prepare colloidally stableGM-E-BDD microgel dispersions it was necessary to use a higher pH forthe functionalisation to avoid aggregation during the process. Therelatively high GMA content for the GM-E-BDD microgels did not decreasethe extent of swelling as judged by the Q values at pH=8 (Table 1).

Example 1 Cross-Linking of Microgel Particles by Formation of anInterpenetrating Polymer Network

Two methods were used:

Method A: In this method the microgel was added first. Typically, thesystem was prepared using 10 wt. % microgel (methods 1 or 2) and 10 wt.% PEGDMA550 stock dispersion. In that case a mixture of 0.2 ml ofammonium persulfate solution (10 wt. % in water), 0.5 ml of aqueous 2 MNaOH was added to a mixture of 2.5 ml of microgel (16 wt. %), 0.36 ml ofPEGDMA550 and 0.44 ml of DI water using stirring. The final weak gellike mixture was held in a water bath and allowed to react at thedesired temperature.

Method B: Cross-linker added first. In this case 2.5 ml of microgel (16wt. %) was added to a pre-prepared mixture of 0.2 ml of ammoniumpersulfate solution (10 wt. % in water), 0.5 ml of aqueous 2 M NaOH,0.36 ml of PEGDMA550 and 0.44 ml of DI water by stirring. Before themicrogel was added the mixture of all of the other materials wereallowed to mix for half a minute. The final liquid like mixture was heldin a water bath.

Characterisation

(i) Effect of Microgel 1 Concentration on Strain Dependent ElasticModulus (G) and tan δ (=G″/ G′) [Note that G″ is the Loss Modulus].

The cross-linked microgel was prepared using Example 1, Method A. Thedispersions contained 10 wt. % PEGDMA (molar mass was 550 g/mol).

Dynamic rheology measurements were performed using a TA instrument AR G2temperature-controlled rheometer with an environmental chamber. Theresults are shown in FIG. 6.

(ii) Variation of (a) G′ and (b) tan δ with Strain for Cross-LinkedMicrogels Prepared Using Microgel 2A and PEGDMA550.

The Microgel and PEGDMA concentrations used were 10 wt. % each. In thiscase in-situ cross-linking method was performed within the geometry ofthe rheometer prior to measurement. The instrument was described above.The preparation was done according to Example 1, Method B. The resultsare shown in FIG. 7.

(iii) Effect of Microgel 1 Concentration on Strain Dependent ElasticModulus (G) and tan δ (=G″/ G) [Note that G″ is the Loss Modulus]

The cross-linked microgel 1 particles were prepared using Example 1,Method B. The dispersions contained 10 wt. % PEGDMA550 (molar mass was550 g/mol). The instrument was the same as that described above in (i)above. The results are shown in FIG. 8.

Example 2 Cross-Linking of Microgel Particles by Formation of anInterpenetrating Polymer Network and Study of PrecursorMicrogel/Cross-Linker Networks

For a total hydrogel composite containing a total polymer volumefraction (φ_(Tot)) of 0.20, with a microgel volume fraction with respectto total polymer (Φ_(μ)*) of 0.5, a mixture of 0.02 wt. % ammoniumpersulfate was combined with an appropriate amount of sodium hydroxide(Aldrich, 98%) to give a final solution pH of 7.4. To this thecrosslinking monomer (X) was added, with the same mole percent aspoly(ethylene glycol) dimethacrylate (PEGD550). PEGD (average M_(n) 550,Aldrich) was then dissolved in this solution, to which 10 wt % ofmicrogel (Microgel μ-BDD—obtained from Method 2 above) was addeddropwise whilst mixing using a vortex mixer. The dispersion was thenheated at 50° C. for 16 hours.

In this study we use constant molar concentrations of added crosslinkingco-monomer. That is, for EGD and the different PEGD crosslinkingmonomers a constant crosslinking monomer mol. % (x_(X)) is added whendata are compared. The value of x_(x) is the mol. % of X present withrespect to all of the monomers present in the μ-BDD/H-X composite.Therefore, the mol. % of monomers present within the microgel is100−x_(x). The consequence of this is that there are different volumefraction of crosslinking monomer present.

The microgels are identified as μ-BDD where μ indicates microgel and BDDidentifies the crosslinking monomer used (i.e. poly(EA/MAA/BDDA) asobtained from Method 2). When X is polymerised to form a hydrogelnetwork phase this is identified as H-X. The systems (dispersions orgels) contain either μ-BDD and X: (i.e., μ-BDD/X) or μ-BDD and H-X:(μ-BDD/H-X). The microgel polymer volume fraction in the μ-BDD/X orμ-BDD/H-X mixture with respect to the polymer and monomer or hydrogelpresent is φ_(μ-BDD). The crosslinking monomer volume fraction presentis φ_(X). In the microgel/hydrogel composites the volume fraction of thehydrogel polymer (formed by X) is φ_(H-X). In both the μ-BDD/Xdispersions or μ-BDD/H-X composite gels the total volume fraction ofpolymer and crosslinking monomer is φ_(Tot). The following equationsapply.

For the μ-BDD/X dispersions:

φ_(Tot)=φ_(μ-BDD)+φ_(X)   (4)

For the μ-BDD/H-X composite gels:

φ_(Tot)=φ_(μ-BDD)+φ_(H-X)   (5)

The volume fraction of microgel with respect to microgel and monomer (orhydrogel) is Φ_(μ).

$\begin{matrix}{\Phi_{\mu} = \frac{\varphi_{\mu - {BDD}}}{\varphi_{Tot}}} & (6)\end{matrix}$

The first part of the study investigated the properties of the μ-BDD/Xmixtures. These mixtures were used to prepare the hydrogels. It wasimportant to investigate any changes in microgel properties caused byaddition of the monomers. In order to provide good controls the mixtureswere heated for the same period of time as used for hydrogel formation;however, APS was not added.

The μ-BDD/X mixtures were prepared initially using a range of φ_(μ-BDD)values. It was found that the behaviour of the mixtures was dependent onthe molecular weight of X. If EGD was used then the dispersions remainedphysical gels over φ_(μ-BDD) values from 0.05 to 0.15. However, ifPEG550 was used then the physical gels changed to fluids (as judged bytube inversion) when φ_(μ-BDD) was less than 0.125. Selected images areshown in FIG. 9C. Drawing upon an earlier study which investigatedrelated microgels in the presence of linear PEG homopolymers⁹ thisbehaviour is attributed to osmotic deswelling of the microgels cause byexclusion of the higher molecular weight PEGD550. The lower molecularweight EGD was able to migrate into the interior of the microgelparticles and did not cause particle collapse. It is also possible tosee evidence of this visually through turbidity changes (see FIG. 9C).FIG. 9C shows images of selected concentrated dispersions. The valuesfor φ_(μ-BDD) and x_(X) are shown. The pH was 7.4. The turbidity appearsindependent of φ_(μ-BDD) for the μ-BDD/EGD dispersions. However, itdecreases markedly for the μ-BDD/PEGD dispersions with increasingφ_(μ-BDD). At high φ_(μ-BDD) values there is not enough excluded PEGD550to de-swell the particles.

We probed the effect of PEGD molecular weight using dynamic rheologymeasurements in order to investigate evidence of a cut off value.

FIG. 6C shows variation of (a) G′ and (b) tan δ with strain for μ-BDD/Xdispersions. The molecular weight of X is shown in the legend. (c) Showsthe values for G′ and tan δ measured at 1% strain. (d) Shows thevariation of the yield strain with molecular weight of X. In all casesφ_(μ-BDD)=0.1 and pH=7.4. The value for x_(X) used was 15 mol. %

FIG. 6C(a)-(c) show clearly that G′ falls and tan δ increases withmolecular weight of X. In our experience a G′ of about 100 Pa isrequired for a gel to survive tube inversion. Consequently, therheological data are consistent with the images of the tubes shownabove. It appears from these data that the critical PEGD molecularweight for complete exclusion from the microgel is between 550 and 750g/mol. This corresponds to 9-13 EO units. Under these conditions themicrogel particles are sufficiently collapsed that the physical gels arenot strong enough to support their own weight when subjected to tubeinversion (FIG. 9C).

The yield strain (γ*) is defined here as the strain at which G′ falls to95% of its value at 1% strain (Chougnet, A.; Audibert, A.; Moan, M.Rheol. Acta 2007, 46, 793). This marks the transition to networkbreakdown. It can be seen from FIG. 6C(d) that there is considerablevariation with molecular weight of X for these values. Generally, thephysical gels are brittle materials and γ* does not seem to be relatedto G′.

Characterisation

(i) Volume Swelling Ratio (Q) for Cross-Linked Microgels FormedAccording to Example 2 Measured after 7 Days as a Function of μ-BDDVolume Fraction.

The swelling behaviour was investigated for the μ-BDD/H-PEGD550 hydrogelcomposites at pH=7.4 after 7 days.

FIG. 8C-1 shows μ-BDD/H-PEGD550 swelling ratio and sol fraction as afunction of Φ_(μ)*. The inserts show selected μ-BDD/H-PEGD550 gels. Thecomposites were prepared using Φ_(Tot)=0.2. Note thatΦ_(μ-BDD)=Φ_(μ)*×Φ_(Tot). The data and images were pH 7.4. Q for theμ-BDD microgel (Table 1) is shown for comparison. The images and datawere obtained after 7 days.

The gels were prepared using φ_(Tot)=0.2 and a range of Φ_(μ) values.The Q and SF values were constant until Φ_(μ) exceeded 0.25. At higherΦ_(μ) values Q increased while SF decreased. This is attributed to adecrease in the PEGD550 matrix which had a substantial SF (of about 0.5)and a lower average molecular weight between crosslinks. The microgelparticles had a higher Q value than poly(PEGD) because they onlycontained about 0.5 mol. % of BDD. This can be seen by comparing thedata points at φ_(Tot)=0 and 1.0, respectively, for poly(PEGD550) andμ-BDD microgel particles.

The abrupt change in behaviour at Φ_(μ)=0.25 is interesting. It may bethat this is where a percolated network of microgel particles within theH-PEGD550 matrix first forms. Such as network would be expected toreduce the overall effectiveness of the H-PEGD550 crosslinked phase toconstrain the microgel particles. At Φ_(μ) values greater than or equalto about 0.63 the gel re-dispersed. This value for Φ_(μ) is very closeto the packing volume fraction for a hexagonally close packed system ofmonodisperse spheres (0.64). It is also in the region of volumefractions where a trapped glass is expected (Debord, S. B.; Lyon, L. A.J. Phys. Chem. B. 2003, 107, 2927). It would be reasonable to expect theencapsulating H-PEGD550 phase to become fragmented (non-continuous)under these conditions. The point at which μ-BDD/H-PEGD550 re-disperses(φ_(M)≧0.63) corresponds to the point at which the parent μ-BDD/PEGD550mixtures form physical gels (FIG. 9C). This shows that re-dispersion isdue to the inability of PEGD550 to form a continuous membrane throughoutthe dispersion.

(ii) Volume Swelling Ratio (Q) for Cross-Linked Microgels FormedAccording to Example 2 Measured After 7 Days as a Function of theMolecular Weight of X (Cross-Linker)

The effect molecular weight of X on the swelling was also investigated(See FIG. 8C-2).

FIG. 8C-2 shows the effect of MW on swelling ratio and sol fraction forμ-BDD/H-X hydrogel composites. The composites were prepared usingφ_(μ-BDD)=0.10 and x_(X)=15 mol. %. The gels were then equilibrated atpH=7.4 for 7 days prior to measurement.

It appears that Q increases and SF decreases with MW of the crosslinkingmonomer. The increase of Q is expected from an increase in the averagemolecular weight between crosslinks. It is not clear why the SFdecreases with MW for these systems at this stage although entanglementsmay be expected to become more important as the molar mass of Xincreases.

(iii) Volume Swelling Ratio (Q) for Cross-Linked Microgels FormedAccording to Example 2 Measured After 7 Days as a Function of pH

The μ-BDD/H-PEGD550 and μ-BDD/H-EGD gels were allowed to reach swellingequilibrium. The respective Q values are shown in FIG. 8C-3.

FIG. 8C-3 shows variation of Q for hydrogel composites with pH. Thecomposites were prepared at pH 7.4, φ⁻ _(μBDD)=0.10 and x_(X)=15 mol. %,placed in 0.1M buffer and allowed to equilibrate for 7 days. Data forμ-BDD obtained from PCS are also shown.

Generally, there is agreement between the Q values for the gels and theμ-BDD microgel particles. However, there is considerable scatter for thegel data which prevents a more detailed analysis.

(iv) Effect of Microgel μ-BDD Concentration in Example 2 on StrainDependent Elastic Modulus (G) and tan δ (=G″/G′) [Note that G″ is theLoss Modulus]

Composite hydrogels (μ-BDD/H-X) were prepared by adding initiator (APS)to the concentrated dispersions discussed above under Example 10. Theresult of the crosslinking was gels that were more resilient tore-dispersion (later). There was also an increase in the modulus. Theincrease was most pronounced for the μ-BDD/H-PEGD550 system.

FIG. 7C shows the effect of variation of φ_(μ-BDD) for μ-BDD/H-EGD andμ-BDD/H-PEGD550 hydrogel composites. Selected data for the respectiveμ-BDD/EGD dispersions are also shown for comparison. The values forx_(X) are also shown. All systems were measured at pH 7.4. These dataillustrate the gap in G′ values that becomes increasingly pronounced inthe region φ_(μ-BDD) of about 0.10 to 0.17. This indicates an optimumrange for osmotic de-swelling of the μ-BDD particles in the presence ofPEGD550 before crosslinking.

(v) Effect of Molecular Weight of the Crosslinking Monomer (X) on theMechanical Properties of Composite Gels of Example 2

The effect of crosslinking monomer molecular weight on the mechanicalproperties of composite gels prepared using φ⁻ _(μBDD)=0.1 and x_(X)=15mol. % was investigated in more detail. The results from these data areshown in strain amplitude studies were performed.

FIG. 8C-4 shows data for μ-BDD/H-x composite gels: Effect of molecularweight of X on (a) G′, (b) tan δ and (c) γ*. The data were obtainedusing φ_(μ-BDD)=0.10, and x_(X)=15 mol %. The data for (a) and (b) weremeasured using 1% strain and 1 Hz. Note that the point at MW of 0corresponds to φ⁻ _(μBDD)=0.20. These data show evidence for a clearchange in G′ and γ* with MW of X. It can be seen that the MW of 330g/mol marks a change in the rate of decrease of G′ with MW and also amaximum value for γ* (of 19%). This is an indication of a relativelylarge molar mass between crosslinking points at the particle periphery.The data also show (FIG. 8C-4( b)) that there is an abrupt increase intan δ at high MW values. This is an indication of increasing dissipationdue to a weak network surrounding the de-swollen particles.

Comparison of the data shown in FIGS. 6C (see Example 10) and 8C-4 showsthat the greatest increase for G′ upon composite formation occurs when Xis excluded from microgel interior; in that case the fluid (mixture)changes to a solid gel. There is not a great deal of difference for theG′ values for the low molar mass crosslinking monomers.

(vi) Effect of φ_(μ-BDD) on G′, tan δ and γ* of Products of Example 2

The effect of φ_(μ-BDD) on G′, tan δ and γ* were investigated and thedata appear in FIG. 8C-5.

FIG. 8C-5 shows the effect of φ_(μ-BDD) on (a) G′, (b) tan δ and (c) γ*for μ-BDD/H-EGD and μ-BDD/H-PEGD550 hydrogel composites. The value forx_(X) was 15 mol. % in each case.

It can be seen that a minimum φ_(μ-BDD) of about 0.05 is required forthe μ-BDD/H-PEGD550 systems in order to form a gel with significantelasticity and tan δ<1.0. In the case of μ-BDD/H-EGD the minimum is muchlower (less than 0.025). The exponential relationships (FIG. 8C-5( a))imply tunability through control of φ⁻ _(μBDD). It is interesting thatfor both systems there appears to be a φ_(μ-BDD) at which the G′ and tanδ values become identical and this is 0.125. The rheological propertiesof the composites are identical which implies that there is nodifference between the load distribution within each type of network atthat value of φ_(μ-BDD).

The above data demonstrates considerable tunability for the composites.The greatest changes upon covalent crosslinking occur for theμ-BDD/H-PEGD550 gels. When φ_(μ-BDD)=0.1, they change from a fluid to agel. This indicates good potential for an injectable dispersion.

(vii) Effect of pH on Strain Dependent Elastic Modulus (G′) and tan δ(=G″/G′) [Note that G″ is the Loss Modulus] for Example 2

The rheological properties of the equilibrium swollen gels were alsoinvestigated.

FIG. 8C-6 shows variation of (a) G′ and (b) tan δ with pH for μ-BDD/Xcomposite hydrogels. These were the same gels used for the swellingexperiments shown in FIG. 8C-3.

It can be seen that in the physiological pH range the composite gelshave low G′ values. They were also quite brittle with γ* values lessthan 5%. Both of these effects can be attributed to highly swollenchains. The Q values are in the vicinity of 30 to 40 from FIG. 8C-3 atthese pH ranges (7 to 7.4). This corresponds to a φ_(P) value of only0.02 to 0.03. Therefore, in the fully swollen state these composites areweak gels. However, the data from FIG. 8C-5 shows that much more elasticgels can be achieved by limiting swelling to a range where the φ_(P)values are larger.

Conclusions

The type of hydrogel composite that is obtained depends on the MW of theadded crosslinking monomer. If the MW is smaller than the exclusionlimit (ca. 550 g/mol) then it penetrates the swollen microgels andreinforces the physical gels to produce network threaded microgels. Ifthe MW is greater than this value the monomer is excluded and crosslinksaround the microgel particles, encapsulating them, to form amicrogel-reinforced hydrogel. In the latter case the excludedcrosslinking monomer caused deswelling and this resulted in a fluidrather than a physical gel. The μ-BDD/X fluid changed to a gel uponcrosslinking. This is an advantageous result from the viewpoint ofpotential application because the latter systems are injectable prior togelation. The work has also demonstrated that the mechanical propertiesof the hydrogel composites can be tuned by the composition used fortheir preparation. The modulus values obtained in this work (1000-30,000Pa) match those of a range of soft tissues in the body. Further, theability to tune the modulus values suggests that the mechanicalproperties of these hydrogel composites will be suitable for applicationin intervertebral disc repair.

Example 3 Cross-Linking of the Vinyl-Grafted Microgel Particles

2.5 ml of poly(MMA/MAA/EGDMA)-GMA microgel (16 wt. %) was added to amixture of 0.2 ml of ammonium persulfate solution (10 wt. % in water),0.5 ml of aqueous 2 M NaOH and 0.8 ml of DI water by stirring. The finalpH was maintained between 7.5 and 8.5. The dispersion was heated to thedesired temperature. In the case of preparations conducted at 37° C.,TEMED was added at concentrations between (2 and 50 mM).

FIG. 9 shows a photographic image of: (a) a Microgel 2B dispersion (atpH=7.3); (b) a cross-linked 2BG microgel; and (c) a cross-linked 2BGmicrogel that has swollen in neutral pH water. For each gel shown inFIG. 9, the initial microgel concentration corresponded to 7 wt. %. Thegels were prepared using the procedure outlined in Example 2 above at atemperature of 50° C., and in the absence of added TEMED.

FIG. 10 shows scanning electron micrograph images of: (a) a cross-linked2BG microgel and (b) a non-cross-linked microgel dispersion (Microgel2B), both at the same particle concentration (10 wt. %). SEM image wasobtained using a Philips FEGSEM instrument. Sample (a) was preparedusing the method described in Example 2 above and sample (b) wasprepared using the procedure described in Method 1. In the case ofsample (a) TEMED was not added and the reaction was performed at 50° C.

Characterisation

(i) Volume Swelling Ratio (q_(gel)) for Cross-Linked Gels of Microgel2BG Measured After 7 Days as a Function of pH.

Data measured in a buffered phosphate buffered saline solution (PBS) arealso shown. The value for q_(gel) was measured using the ratio of thegel volume at a given pH to the volume of the water-free (dry) gel. Thiswas done gravimetrically after blotting excess water from the swollengel using tissue paper.

The double cross-linked microgel was prepared using heating at 50° C. asdescribed for Example 2. However, in this case TEMED was not added.

The results are shown in FIG. 11.

(ii) Effect of Microgel 2BG Particle Concentration Used DuringCross-Linking on (a) G′ and (b) tan δ as a Function of Strain.

The double cross-linked microgel was prepared using heating at 50° C.using Example 2 in the absence of added TEMED. The values of γ* weredetermined as described above. The instrumentation for rheology was alsodescribed above.

The results are shown in FIG. 13. The volume fraction of polymer usedduring preparation is shown in the legend. [Multiply by a factor of 100to convert to wt. %] The variation of G′, γ* and tan δ with volumefraction of polymer are shown in (c) and (d).

The pH for these data was 7.8. For these measurements, and the othersgiven in the examples, the oscillation frequency was used 6.3 rad/sunless otherwise stated.

(iii) Variation of (a) G′ and (b) tan δ with Strain for Cross-LinkedMicrogels Prepared.

The data were obtained using Microgel 2BG (open diamonds) and 3G (closeddiamonds). The double cross-linking was performed at 37° C. usingammonium persulfate (22 mM) and 10 wt. % of the microgel for 17 h usingExample 2 in the absence of added TEMED. The value for γ* for doublycross-linked 3G is in the vicinity of 500%. For these experiments thehydrogel was prepared in-situ within the rheometer just prior tomeasurements occurring using Example 2. The results are shown in FIG.14.

(iv) Variation of (a) G′ and (b) tan δ with Strain for DoublyCross-Linked Microgels Prepared from Microgel 2BG and TEMED (7.98 mM).

The cross-linking was performed at 37° C. The reaction time is shown.The estimated values for g* is 195% for the system after 120 min ofreaction. The system was prepared using 10 wt. % Microgel 2BG.

This was measured after situ cross-linking within the rheometer usingthe procedure of Example 2. The results are shown in FIG. 15.

Example 4 Cross-Linking of the Vinyl-Grafted Microgel Particles

Generally, the doubly cross-linked microgel particle composition (DXmicrogel) was prepared using φ_(P)=0.10, pH=7.8, 22 mM of APS and areaction temperature of 50° C. The GM-functionalised microgel (e.g.Microgel GM-M-EGD of Method 3A) was added to the NaOH/APS solution withvigorously mixing for about 5 minutes to form a physical gel at roomtemperature. After fully mixing the physically gelled dispersion washeated at 50° C. for 8 h, and was allowed to react to yield DX GM-M-EGD.

For measurements performed using rheology the crosslinking was conductedin-situ within the rheometer for at least 1 h prior to commencing themeasurements. For the larger samples prepared for swelling measurementsthe reaction time was 8 h.

FIG. 9A shows an SEM photographic image of: (a) Microgel M-EGD (ofMethod 1A); (b) Microgel GM-M-EGD (of Method 3A). The particles arespherical although the polydispersities are significant. Table 2 showsthat the number-average particles sizes for these two microgels wereabout 130 nm and not significantly affected by functionalisation.Comparison of the diameters measured by SEM and also PCS (at pH=4) showsthey are similar. This indicates that there was negligible aggregationof the particles in dispersion.

TABLE 2 Mol. % Mol. % d_(n(sem))/ d_(h(4)) ^(c)/ d_(h(8)) ^(c)/ CodeMAA^(a) GMA^(a) nm (CV)^(b) nm nm Q₍₈₎ ^(d) pK_(a) ^(e) M-EGD 35.9 — 131(14) 139 232 4.7 7.4 GM-M-EGD 34.1 1.8 133 (20) 131 323 15 7.1 GM(H)-M-35.9 5.8 133 315 13 6.0 EGD ^(a)Determined from potentiometric titrationdata. The mol. % GMA was determined from the difference in the mol. %MAA in the microgel before and after functionalisation.^(b)Number-average diameters determined from SEM images. The number inbrackets is the coefficient of variation. ^(c)Hydrodynamic diameter atpH values of 4 and 8. ^(d)Swelling ratio calculated using d_(h(8)) andd₍₄₎ values for the parent microgel according to equation (1) - seetext. ^(e)Apparent pK_(a) values. These are the pH values correspondingto 50% neutralisation.

FIG. 10A shows scanning electron micrograph images of freeze-driedsamples of: (a) DX GM-M-EGD and (b) M-EGD (i.e. . . . The sample wasprepared using φ_(P)=0.10 and pH=7.8. The insets for (a) and (b) show apictures of a free-standing DX GM-M-EGD microgel and a physicallygelled, M-EGD dispersion, respectively. SEM image was obtained using aPhilips FEGSEM instrument. Sample (a) was prepared using the methoddescribed in Example 2A above and sample (b) was prepared using theprocedure described in Method 1A. Freeze-drying has a tendency toproduce micrometer-sized voids as a consequence of ice formation duringsample immersion in liquid nitrogen.

Nevertheless, it was found that features on the scale of individualparticles were less common with the DX gels (FIG. 10A(a)) compared tothe parent SX M-EGD gel (FIG. 10A(b)), which may indicate a greaterextent of inter-particle intepenetration. The GM-M-EGD particles had agreater tendency to swell than the M-EGD particles (see below).

Characterisation (i) Volume Swelling Ratio (Q) for Cross-Linked Gels ofMicrogel DX GM-M-EGD as Prepared in Example 4 After 8 Days as a Functionof pH.

This double cross-linked microgel was DX GM-M-EGD as prepared in Example2A.

Physical measurements were conducted as described above.

In the following study the mechanical properties of DX microgels thatwere allowed to reach swelling equilibrium were studied. We usedconditions just below the critical φ_(p) value of 0.10 because this wasa more stringent test of whether DX microgels could in fact surviveswelling without disintegration. Furthermore, the SX gel hadsufficiently low G′ values that they were fluid when sheared by tubeinversion. This means that these mixtures would be injectable through anarrow gauge syringe. That would be advantageous for soft tissue repairif low temperature crosslinking was used.

The DX microgels swelled strongly in buffer or water. The DX GM-M-EGDmicrogel swelled so strongly in water that it fragmented macroscopicallyafter several days. This shows that the inter-particle crosslinking wasnot sufficiently strong to withstand the swelling pressure within theparticles. If the DX microgel was placed in buffer solutions (ionicstrengths of ca. 0.1 M) they gave robust gels that did not fragment. Thehigh ionic strength reduced the extent of swelling and shows theimportance of electrostatic repulsion in the swelling of these DXmicrogels.

The Q_(DX) values for the DX microgels was measured as a function oftime (FIG. 12A).

FIG. 12A. shows (a) Swelling ratios as a function of time for DXGM-M-EGD prepared using φ_(p)=0.08. The lines are guides to the eye. (b)Variation of swelling ratios measured after 8 days with pH for the DXGM-M-EGD (solid squares), DX GM-EGD(PBS) (open squares) and SX GM-M-EGD(open diamonds—this is product of method 3A, parent microgel) microgels.The initial pH was 7.8. The equations used for these data were (1) and(2).

The swelling was slow due to the close packed nature of these doublycrosslinked gels. We hypothesised that a close packed arrangement ofparticles was a requirement for preparing load supporting gels. The DXmicrogels of Cho et al.⁸ were formed by a different process (attractiveinteractions) and had much faster swelling kinetics due to their moreopen morphology. The data shown in FIG. 12A reveals a significantdifference between the Q_(DX) (values and the Q values for the SXGM-M-EGD microgels. The cause of the increased swelling for the DXGM-M-EGD microgels must be increased swelling between particles, i.e., alower inter-particle crosslink density. This would suggest a higherM_(c) at the particle periphery (linking particles) than in the particleinterior. This would seem reasonable given (a) the low GMAfunctionalisation (1.8 mol. %) for the DX GM-M-EGD microgel and (b) thefragmentation that occurred for this system when swollen by water.

FIG. 12A.1 shows swelling ratios for (a) DX GM-M-EGD and DX GM(H)-M-EGDmicrogels as well as (b) DX GM-E-BDD microgel as a function of timemeasured at different pH values. The lines are guides to the eye. The DXmicrogels swelled in buffer solutions when the pH was greater than orequal to 7.4 and gave robust gels that did not fragment. The DXmicrogels required at least 1 day to reach full swelling. This issupport for a space-filling morphology that is free of significantmicroporosity and is consistent with the SEM images (FIGS. 10A). Thisslower swelling is different to the rapid swelling (minutes) observedfor DX microgels prepared by a bridging aggregation. We suggest that thepore-free morphology (on the micrometer scale) of our DX microgelscontributes to their high values of G′.

As a final study the rheological behaviours of the equilibrium swollenDX gels were probed (FIG. 12A-1). We selected DX microgels prepared atfairly low φ_(p) values in order to obtain high swelling ratios. Strainamplitude data appear in FIG. 12A-2. The gels used for this figure werethose from FIG. 12A.

FIG. 12A-1 shows (a) Variation of G′ and tan δ with pH for DX GM-M-EGDmicrogels. Triangles and diamonds are G′ and tan δ, respectively. Theclosed symbols show data points obtained using PBS. (b) Variation of γ*with pH for the DX microges. The double crosslinking was performed usingφ_(p)=0.08 (pH=7.8) and the samples were swollen at the pH values shownfor 8 days prior to measurement.

FIG. 12A-2 shows variation of (a) G′ and (b) tan δ with strain for DXGM-M-EGD microgels after swelling at different pH values (or inphosphate buffered saline, PBS) for 8 days. The frequency used was 1 Hz.The swelling ratios for theses DX microgels are shown in FIG. 12A-1.

Interestingly, the G′ values reached ca. 10⁶ Pa at the lowest pH (of3.8). This process of enhancing G′ began once the pH was decreased toless than or equal to 5.8. A decrease in the value for M_(c) occurs asthe pH decreases. Hydrogen bonding between nearby RCOOH groups may alsocontribute to decreasing M_(c). At pH=3.8 the DX microgel was brittleand γ* value decreased to about 2% (FIG. 10( b)). At pH=7.4 these DXmicrogels contain about 98% water at swelling equilibrium and have amodulus of about 10³ Pa with γ* of 8.4%. These values may suitable forpotential application as injectable dispersions for soft tissue repair.Tunability of these properties should be achievable through the O_(p)value used during DX microgel formation and also through monomerselection.

(ii) Variation of (a) G′ and (b) tan δ with Strain for Cross-LinkedMicrogels Prepared.

The following equation, which originates from rubber elasticity theory,can be used to describe the modulus of a network^(20,21).

$\begin{matrix}{G \cong \frac{\rho \; {RT}}{M_{c}}} & (7)\end{matrix}$

For equation (7) G is the shear modulus, ρ is the density of thepolymer, R and T have their usual meanings and M_(c) is the numberaverage molecular weight between crosslinks. The latter is the molecularweight of the elastically effective chains. The value for G′ shouldincrease with decreasing M_(c). This will have two contributions from DXmicrogels; intra-particle and inter-particle crosslinks.

Dynamic rheology was used to investigate the mechanical properties ofthe DX gels. Strain amplitude data are shown in FIG. 14A-1. These dataconsist of the two parent SX physical gels (M-EGD or E-BDD and GM-M-EGDor GM-E-BDD) and the respective DX microgels (DX GM-M-EGD or DXGM-E-BDD). FIG. 14A-1 shows a comparison of strain amplitudemeasurements for DX and SX microgels based on M-EGD ((a) and (b)) andE-BDD ((c) and (d)). The data were obtained at the same particle volumefraction (0.10) and pH (7.8). The frequency used was 1 Hz. The arrows in(c) and (d) show the abrupt change in the G′ and tan δ values thatoccurred.

It can be seen that a slight increase in G′ occurred for both of the GMfunctionalised physical gels compared to the respective parent microgel.This is attributed to the greater hydrophobicity of GM functionalisedmicrogels. Importantly, there is a major increase in G′ upon doublecrosslinking. Moreover, the tendencies for the G′ values (FIG. (14A-1(a)and (c)) to decrease and tan δ values (FIG. 14A-1( b) and (d)) toincrease at high strain are greatly diminished as a consequence ofdouble crosslinking. Both of these behaviours are indirect evidence forinter-particle crosslinking.

It can be seen from FIG. 14A-1( d) that the tan δ values areexceptionally low for DX GM-E-BDD microgel with an average tan δ of ca.0.01. That means that the energy loss from dissipation was less than 1%of the energy stored in this DX microgel network. The mechanicalproperties of the DX GM-E-BDD microgel were almost completely elastic.Interestingly, this DX microgel does not show strain-induced networkbreakdown until the strain reaches 50% (FIG. 14A-1( c)). All of themicrogels studied here generally satisfy one key criterion to beconsidered as gels^(22,23), i.e., tan δ<1.0. However, for the DXGM-E-BDD microgel tan δ is also independent of frequency, which is asecond criterion that many gels do not satisfy.

FIG. 14A-.1.1 shows strain amplitude ((a) and (b)) and frequency sweep((c) and (d)) dynamic rheology data for DX GM(H)-M-EGD, DX GM-E-BDD andDX GM-M-EGD microgels, where the DX microgels were prepared atφ_(p)=0.10 and pH=7.8.

It was expected that M_(c) would be inversely proportional to the degreeof functionalisation. Therefore, G′ was expected to increase withfunctionalisation according to equation (7). This was investigated usingDX GM-M-EGD and DX GM(H)-M-EGD microgels. The latter had a higher GMfunctionalisation (5.8 mol. % cf. 1.8 mol. %). Strain amplitude andfrequency sweep rheology data for the DX GM(H)-M-EGD and GM-M-EGDmicrogels are shown in FIG. 14A-1.1. Comparing the data shown in FIG.14A-1.1 (a) and (b) it is clear that the G′ values for DX GM(H)-M-EGDare much higher than those for DX GM-M-EGD. At a strain of 1% the valueof G′ for DX GM(H)-M-EGD was 18,800 Pa, and is six times the value forDX GM-M-EGD. This is a high modulus for a gel that only occupies 10 vol.% of the total volume. Furthermore, the value for tan δ decreased to0.045. The frequency dependence of G′ and also tan δ greatly decreased(FIG. 14A-1.1 (c) and (d)). The DX GM(H)-M-EGD microgel is less ductileas tan δ increases more at a lower strain (FIG. 14A-1.1 (b)). The valueof γ* is 8.0%. All of these changes are indicative of a smaller M_(c)value as a consequence of a higher degree of GM functionalisation for DXGM(H)-M-EGD. The changes for G′ and γ* for this system generally matchwhat is expected for a conventional hydrogel and we attribute thesechanges to increased inter-particle crosslinking. These data show thatthe modulus and ductility for the DX GM-M-EGD microgels are tuneableusing the extent of GM functionalisation.

(iii) Variation of (a) G′ and tan δ as Well as (b) γ* with MicrogelParticle Volume Fraction for Doubly Cross-Linked Microgels of Example 4

The effect of GM-M-EGD volume fraction used during double crosslinkingwas also investigated.

FIG. 16A shows (a) Effect of GM-M-EGD volume fraction used during doublecrosslinking on G′ and tan δ. (b) The variation of γ* with volumefraction of polymer. The pH used to obtain these data was 7.8. A strainand frequency of 1% and 1 Hz was used to obtain the data shown in (a).

The data show an increase of G′ with φ_(p). Furthermore, tan δ increaseswith φ_(p) which suggests an increase in dissipation with high microgelconcentrations. FIG. 16A(a) shows an increase for G′ when φ_(p) reaches0.10. FIG. 16A(b) shows that that γ* increases strongly when φ_(p)reaches 0.10. These data suggest a critical φ_(p) value of about 0.10where the inter-particle crosslinking becomes increasingly pronounced.Presumably, higher φ_(p) values result in more extensiveinterpenetration at the periphery of the particles. These data show thatthe G′ of the DX microgel can be readily tuned simply by using microgelparticle concentration.

(iv) Variation of (a) G′ and tan δ as Well as (b) γ* with pH Used DuringCross-Linking in Example 4.

The effect of pH used during double crosslinking was investigated. Aparticle concentration of φ_(p)=0.10 was used for these experiments. Wefirst consider data from strain amplitude experiments (FIG. 14A-2).

FIG. 14A-2 shows strain amplitude data for DX GM-M-EGD microgelsprepared and measured at different pH values (shown in legend). Thevalue for φ_(p) was 0.10. The data plotted were obtained using 1 Hz.

It can be seen that G′ does not change significantly with strain (γ)over the strain range of 0.1 to 10% for pH values between 7.3 and 8.3.It does, however, begin to decrease at higher strain values. These datashow that increasing the pH during preparation (up to 8.3) bothincreases G′ and also the strain required to disrupt the network. Beyonda pH of 8.3 both parameters then decrease. When the pH was greater thanor equal to 9.6 tan δ is greater than 1.0 and the material remainsfluid.

FIG. 14A-3 shows data taken from mechanical spectra (average of strainand frequency sweeps). Note for (c) that only data for the gels (tanδ<1.0) are shown. Data used are 1% strain and 1 Hz. The vertical linesshown in (a) and (b) are the pK_(a) value for GM-M-EGD. The curve in (c)is a guide for the eye.

It can be seen from FIG. 14A-3 that the G′ values increased byapproximately factors of 2 and 7, respectively for the DX GM-M-EGD andGM-E-BDD microgels. This implies that M_(c) decreased by a factor of 2for the DX GM-M-EGD. For the DX GM-E-BDD series M_(c) must havedecreased by a factor of about 7. These data changes are generallyconsistent with equation (3) because the former should have a much lowerM_(c) due to the much higher mol. % of GMA incorporated (Table 1).

The yield strain (γ*) is defined here as the strain at which G′decreases to 95% of its value at 1% strain²⁴. This increases from about8% for the SX gels (FIG. 14A-3( c)) to greater than 30% for the DXGM-M-EGD gel and 55% for the DX GM-E-BDD gel. The increase in this valueis an indication of relatively flexible chains linking the microgelstogether. This could be due to extended chains of the particles at theperiphery which have interpenetrated and crosslinked with chains fromneighbouring particles.

Physical gels must first form in order for a covalently linked microgelnetwork to subsequently form. It can be seen from FIG. 14A-3 that the DXmicrogels with the highest G′ and lowest tan δ values occur at pH valuesbetween 7.3 and 8.3. Indeed, in this pH range (which includesphysiological pH) both G′ and γ* increase with pH used to prepare the DXmicrogels. At higher pH values γ* increased (FIG. 14A-3( c)); however,G′ decreased and tan δ increased to above 1.0 indicating that a fluid ispresent. This is suggestive of an increased molar mass betweencrosslinking points at the particle periphery. At high pH values thisbecomes insufficient to enable gel formation.

The value for γ* will be sensitive to both the M_(c) values within andbetween the microgel particles. The increase for γ* with pH for the DXGM-M-EGD microgels (FIG. 14A-3( c)) suggests that longer elasticallyeffective chains are present at the particle periphery at high pH. AtpH=9.6 the gel was approaching the fluid state (tan δ approaching 1.0).The G′ value was low (96 Pa). However, that system had the highest γ*value of 64%. Moreover, that sample was completely transparent (withoutturbidity). At higher pH values the dispersions did not form physical orcovalently-linked gels. It is likely that electrolyte triggered particlecollapse at high pH (due to screening) reduced the extent of particleinter-penetration to the point where physical gelation did not occur.

Conclusions

In this work a new general method for preparing DX microgels has beendemonstrated. This method uses only functionalised microgels and hasbeen used to prepare two new families of pH responsive DX microgels; DXGM-M-EGD and DX GM-E-BDD. These DX microgels did not re-disperse in 0.1M buffer solutions in the pH range of 3.8 to 9.2 or PBS (0.15 M). Themechanical properties of the as-made DX gels are strongly dependent onpH and also φ_(p) used for preparation. This offers considerableopportunity for tuning these properties for specific applications, e.g.,for soft tissue repair and or load support. This study has shown thatthe modulus and yield strain can be controlled using preparationconditions.

The mechanical properties of the DX microgels appear to be stronglydetermined by those of the parent microgel and also the degree offunctionalisation. The data reveal that high modulus of the physicalgels will lead to high modulus values for the respective DX microgel.However, the extent of increase of the modulus on double crosslinkingincreases with the mol. % of GMA incorporated. The ductility of themicrogels, as judged by γ*, is dependent on inter-particle crosslinkingand increases considerably when a φ_(p) greater than or equal to 0.10 isused during DX preparation. The study has shown that injectabledispersions that can form DX microgels can be achieved using φ_(p)=0.08.If high G′ and yield strains are required, higher φ_(p) values should beused.

The DX GM-E-BDD microgel used in this study to demonstrate generalitywas shown to be remarkable in terms of its elastic properties. It wasfound to behave as a near perfect gel rheologically (tan δ approachingzero and invariant with frequency and high γ*) and exceptionally lowviscous component. This could be an important new gel for soft tissuerepair.

Example 5 Cross-Linking of the Vinyl-Grafted Microgel Particles

Two types of MAA-containing microgels were prepared in this work; M-EGDand E-BDD. The majority of the work was conducted on the M-EGD seriesbecause this system has greatest potential application in soft tissuerepair. M-EGD contains co-mononomers that have been investigated forapplication in bone cement (Hiratani, H.; Alvarez-Lorenzo, C.Biomaterials 2004, 25, 1105.) and contact lenses (Zhang, X. S.; Revell,P. A.; Evans, S. L.; Tuke, M. A.; Gregson, P. J. J. Biomed. Mater. Res.1999, 46, 279). The principle monomer (MMA) is a major component of bonecement. The E-BDD microgels were used to demonstrate the generality ofour new approach.

DX microgels were prepared using a volume fraction of polymer ofφ_(p)=0.10, pH=7.8 and in the presence of 22 mM of APS unless otherwisestated. The AEM-functionalised microgel (Microgel AEM10-M-EGD obtainedfrom Method 3B) was added to a NaOH/APS solution and then vigorouslymixing for about three minutes. After fully mixing the physically gelleddispersion was heated at 50° C. for 8 hours to produce DX AEM-M-EGD.

In the case of microgels investigated using rheology, the DX reactionwas performed for at least 1 hour in the rheometer before the system wascooled to room temperature and the rheology experiments performed.

FIG. 9B shows an SEM photographic image of: (a) Microgel M-EGD (ofMethod 1A); (b) Microgel E-BDD (of Method 2A). Representative SEM imagesfor the microgels are shown in FIG. 1. Spherical particles are evident.

FIG. 10B shows scanning electron micrograph images of air-dried (at roomtemperature) samples of: (a) DX AEM-M-EGD and (b) DX AEM-M-EGD (blown upimage from FIG. 10B(a)). The sample was prepared using φ_(p)([AEM]/[MAA])=0.10 and pH=7.8. SEM image was obtained using a PhilipsFEGSEM instrument.

These samples were air dried (at RT) prior to SEM. They show evidence ofmicrometer and nanometer sized cracks. Interestingly, some microgelparticles can be seen on the surface obtained using high magnification.The similarity of the size of the parent M-EGD microgel particles withthe features surrounded by cracks in FIG. 10B(b) leads to the suggestionthat cracks formed at the interfaces between neighbouring aggregates. Ifconfirmed, this would indicate that the periphery between aggregates isthe weakest point of the DX microgel matrix.

Characterisation

(i) Variation of (a) G′ and (b) tan δ with Strain for Cross-LinkedMicrogels Prepared as Per Example 5.

The DX microgels were prepared from physically gelled microgels.Therefore, the rheological behaviour of the singly crosslinked microgeldispersions were studied at the same pH and φ_(p) values used for doublecrosslinking.

FIG. 14B-1 shows strain amplitude sweeps ((a) and (b)) and frequencysweeps ((c) and (d)) for concentrated SX AEM-M-EGD microgel dispersions.The legends give the [AEM]/[MAA] ratios used for preparation of the AEMfunctionalised microgels. Data for E-BDD microgel are also shown. Themeasurements were made using 1 Hz (strain amplitude) or 1% strain(frequency sweep) using φ_(p)=0.1 and pH=8.4.

From FIG. 14B-1 it can be seen that there is a major difference betweenthe dynamic rheological behaviour for the concentrated M-EGD and E-BDDmicrogels. The former is a weak gel; whereas, the latter has a muchhigher elasticity and yield strain. Following related work (Chougnet,A.; Audibert, A.; Moan, M. Rheol. Acta 2007, 46, 793), the yield strain(γ*) is defined here as the strain at which G′ has fallen to 95% of itsvalue at 1% strain. The variation of G′ and γ* are shown in FIG. 14B-2.

FIG. 14B-2 shows variation of (a) G′ and (b) yield strain with[AEM]/[MAA] ratio for concentrated singly crosslinked microgeldispersions. Data were obtained using φ_(p)=0.1 and pH=8.4. The datapoints for E-BDD was obtained using pH=7.8. The data for (a) wereobtained at 1% strain and 1 Hz.

Based on equation (3) it is suggested that the major increase of G′ forE-BDD compared to M-EGD (factor of 50) is due to the greater overlap andinter-penetration of the microgel. This probably also accounts for themuch greater value for γ* (factor 2.5 higher).

It can also be seen from FIG. 14B-2 that the G′ increases substantiallywith [AEM]/[MAA] ratio used for preparation. The maximum increase isalso a factor of about 50. However, in this case γ* has not increased,but is about the same. This suggests that there is no improvement inoverlap for the gel as a whole. The PCS data (FIG. 2) indicate that noadditional swelling occurred as a result of functionalisation asexpected since there are less MAA groups present in the functionalisedmicrogel. Therefore, it can be concluded that the increases in G′observed for these physical gels upon functionalisation is due to anincreased tendency of the particle aggregates to form physical contactsthrough hydrophobic association. Indeed, it was noted that thefunctionalised microgel dispersions had a tendency to phase separate ifleft for extended periods (month) indicating that aggregation occurred.These data then show that increasing the hydrophobicity of the microgelparticles adds a hydrophobic component to the particle-particle contactsthat occur during double crosslinking. This component dominates the gelbehaviour for the AEM functionalised M-EGD microgels. It can besuggested that these concentrated dispersions consist of swollen,aggregates with a hydrophobic periphery which is rich in AEM groups.

Dynamic rheology measurements were made using the DX microgels asobtained from Example 2.

FIG. 14B-3 shows strain amplitude sweeps ((a) and (b)) and frequencysweeps ((c) and (d)) for DX AEM-M-EGD microgels. The legends give the[AEM]/[MAA] ratios used for preparation. The measurements were madeusing 1 Hz (strain amplitude) or 1% strain (frequency sweep) usingφ_(p)=0.1 and pH=8.4. Data for a doubly crosslinked GM functionalisedmicrogel (DX GM-M-EGD) are also shown for comparison.

The data shown in FIG. 14B-3 reveal that the DX microgels hadconsistently higher G′ values than the SX microgels (Compare to FIG.14B-1). Both the SX and DX microgel systems tended to undergo a majordecrease in G′ and increase in tan δ when the strain exceeded about 10%.The data also permit comparison between our two functionalisationmethods. The maximum G′ value achieved for the DX AEM-M-EGD microgels([AEM/[MAA]=0.5) is approximately a factor of 4.5 that achieved usingthe GMA-functionalisation method. These data suggest that the AEMfunctionalisation method provides DX microgels with higher elasticitythan those achieved using GM functionalisation¹.

The frequency dependent G′ and tan δ data are shown in FIGS. 9( c) and(d). The gradients are very low (especially for tan δ vs. frequency)indicating gel-like behaviour as identified by Winter and Chamboncriteria²⁰⁻²¹. The range of values are the same as for the physical gelscontaining AEM functionalised particles (FIGS. 5( c) and (d)) andconfirms that the DX microgels preserve the low dissipative componentsof their rheological properties.

(ii) Variation of (a) G′ and tan δ as Well as (b) γ* with PreparationConditions for Doubly Cross-Linked Microgels of Example 5

FIG. 14B-4 shows the effect of preparation conditions on the mechanicalproperties of DX microgels. G′ and tan δ values for DX and SX microgelsas a function of [AEM]/[MAA] ratio used to prepare the functionalisedmicrogels are shown in (a) and (b). G′ and γ* values are plotted as afunction of mol. % AEM present within the functionalised microgels in(c) and (d). Values for the DX GM-M-EGD microgel are shown as thehorizontal lines. The data were measured at 1% strain. The data wereobtained using φ_(p)=0.10 and pH=8.4.

The frequency dependent G′ and tan δ data are shown in FIGS. 9( c) and(d). The gradients are very low (especially for tan δ vs. frequency)indicating gel-like behaviour as identified by Winter and Chamboncriteria (Winter, H. H. Polym. Eng. Sci. 1987, 27, 1698; and Winter, H.H.; Chambon, F. J. Rheol. 1986, 30, 367). The range of values are thesame as for the physical gels containing AEM functionalised particles(FIGS. 14B-1( c) and (d)) and confirms that the DX microgels preservethe low dissipative components of their rheological properties.

Data taken from FIG. 14B-3 at a frequency and strain of 1 Hz and 1%,respectively, are shown in FIG. 14B-4. The contrast between DX and SXmicrogels is very clear at higher degrees of functionalisation.Comparison of these data for the DX and respective SX microgels (FIGS.14B-4( a) and (c)) shows clearly that crosslinking provided an increasedG′ in addition to the hydrophobic contribution discussed above. Theincreased G′ values can be attributed to additional covalentcrosslinking from the AEM groups. Moreover, G′ for DX microgels appearsto be proportional to the amount of AEM incorporated when the mol. %AEM(exp) exceeds 3%. The G′ values for the DX microgels of up to 10⁴ Paare respectable values given that φ_(p) is only 0.10. The use of higherφ_(p) values would certainly enable much higher G′ values to beachieved¹.

An interesting point concerns the ability of these DX microgels towithstand strain. Values for γ* are shown in FIG. 14B-4(d). These valuesare between 2.5 and 4.5% and are not significantly different to thosefor the respective SX AEM-M-EGD microgels.

The value of γ* for the DX GM-M-EGD microgel was 6.0%. This was a factorof 2 higher than the values of γ* for the parent microgel (M-EGD) andalso significantly greater than those for the DX AEM-M-EGD microgels.Although the AEM functionalisation method has resulted in a majorincrease in G′ for the M-EGD DX microgels, these gels are inherentlymore brittle. Presumably, this is because the molar mass of theelastically effective chains at the periphery is low. This couldindicate reduced inter-penetration of the peripheries of the aggregatesin the physical gels.

Conclusions

In this study we investigated a new method, involving EDC coupling, forpreparing vinyl functionalised microgels in order to increase themodulus of DX microgels. The titration data indicate that thefunctionalisation proceeds from the exterior of the microgels inwards.Partial aggregation occurred during the functionalisation process. Thisappears to play a role in limiting the maximum degree offunctionalisation that can be achieved to about 12 mol. % in total,i.e., about 1/3^(rd) of the RCOOH groups. Physical gels (SX microgels)formed in concentrated dispersions that had a hydrophobic contributionto their elasticity. Double crosslinking of the partially aggregateddispersions gave gels with high elasticity and this is consistent with arelatively high degree of functionalisaton. The modulus of the DXmicrogels appears to be tunable using the mol. % of AEM incorporated.The results support the suggestion that EDC coupling would increasefunctionalisation and elastic modulus. The mechanical properties ofthese DX microgels can be controlled by their composition. In theprevious work (Liu, R.; Milani, A. H.; Freemont, T. J.; Saunders, B. R.Manuscript submitted to Soft Matter 2011) the maximum G′ achieved was2,800 Pa. An improvement of elastic modulus by about a factor of 4.5compared to the previous method was found for the DX AEM50-M-EGDmicrogel. However, these DX microgels are more brittle, with a yieldstrain that decreased by about a factor of 2. This is most likelybecause of reduced overlap between the aggregates due to the morehydrophobic microgel particle peripheries. Future work will involvewashing the M-EGD microgels with ethyl acetate prior tofunctionalisation in order to increase the swelling and overlap duringdouble crosslinking. We expect the technique to apply well to theAEM-E-BDD microgels and this is currently under being studied. Asuccessful result will show that the technique is widely applicable toRCOOH containing microgels.

Example 6 Cross-Linking of the Vinyl-Grafted Microgel Particles by UVIrradiation

2.5 ml of poly(MMA/MAA/EGDMA)-GMA microgel (16 wt. %) was added to amixture of 0.2 ml of Irgacure 2959 (10 wt. % in ethanol), 0.5 ml ofaqueous 2 M NaOH and 0.8 ml of DI water by stirring. The final pH wasmaintained between 7.5 and 8.5. The dispersion was exposed to UV lightfor 2.5 hours.

Characterisation

(i) Variation of (a) G′ and tan δ as Well as (b) γ* with MicrogelParticle Volume Fraction for Doubly Cross-Linked 2BG Microgel.

The results are shown in FIG. 16. To convert to wt % multiply φ_(p) by100. The double cross-linking was performed at using UV irradiationusing Microgel 2BG using the method described in Example 6.

Example 7 Cross-Linking of Vinyl-Grafted Microgel Particles by Formationof an Interpenetrating Polymer Network

Three methods were used:

Method A: The microgel added first. Typically, the system contained 10wt. % microgel and 10 wt. % PEGDMA550, a mixture of 0.2 ml of ammoniumpersulfate solution (10 wt. % in water), 0.5 ml of aqueous 2 M NaOH wasadded to a mixture of 2.5 ml of poly(MMA/MAA/EGDMA)-GMA microgel (16 wt.%), 0.36 ml of PEGDMA550 and 0.44 ml of DI water by stirring. The finalweak gel like mixture was held in a water bath and allowed to react atthe desired temperature.

Method B: Cross-linker added first. In this case 2.5 ml ofpoly(MMA/MAA/EGDMA)-GMA microgel (16 wt. %) was added to a pre-preparedmixture of 0.2 ml of ammonium persulfate solution (10 wt. % in water),0.5 ml of aqueous 2 M NaOH, 0.36 ml of PEGDMA550 and 0.34 ml of DI waterby stirring. Before the microgel was added the mixture of all of theother materials were mixed for half a minute. The final liquid likemixture was held in a water bath and allowed to react at the desiredtemperature.

When required, accelerator, TEMED, was added to the mixture of ammoniumpersulfate and NaOH solution before microgel or PEGDMA was added withinMethod 6A and Method 6B. The addition of TEMED decreased thecross-linking time and enabled a temperature of 37° C. to be used.

Method C: UV-Irradiation

In the UV-light initiation case 2.5 ml of poly(MMA/MAA/EGDMA)-GMAmicrogel (16 wt. %) was added to a mixture of 0.2 ml of Irgacure 2959solution (10 wt. % in ethanol), 0.5 ml of aqueous 2 M NaOH, 0.36 ml ofPEGDMA550 and 0.44 ml of DI water by stirring. Before the microgel wasadded the mixture of all of the other materials were mixed for half aminute. The final weak gel like mixture was placed under UV light for 3hours

Characterisation

A cross-linked microgel composition of the invention formed by themethod of Example 7, Method B (and in the presence of TEMED) is shown inFIG. 17. The material was prepared using 10 wt % PEGDMA550 and 10 wt. %microgel 2BG. The gel was prepared at 37° C. The scale bar is incentimetres.

Variation of (a) G′ and tan δ as Well as (b) γ* with wt. % of PEGDMA550Used to Prepare Doubly Cross-Linked Microgels.

The total concentration of Microgel 2BG and PEGDMA550 was 20 wt. %. Itshould be noted that the PEGDMA550 only system did not form aspace-filling gel; whereas, the other systems did. The doublycross-linked microgels were prepared using UV cross-linking using theprocess described in Example 4, Method C. The results are shown in FIG.18.

1. The composition, comprising a plurality of microgel particles,wherein adjacent microgel particles are bound together by either (i) acovalent cross-links formed by the reaction of vinyl-containing moietiesgrafted onto the surfaces of the microgel particles; and/or (ii) across-linked polymer network that interpenetrates adjacent microgelparticles and thereby binds the particles together, wherein the polymernetwork is formed by the polymerisation of a water soluble cross-linkingmonomer comprising two or more vinyl groups.
 2. The compositionaccording to claim 1, wherein the microgel particles are pH responsive.3. The composition according to claim 1, wherein the microgel particlescomprise a polymer selected from poly(EA/MAA/EGDMA),poly(MMA/MAA/EGDMA), poly(EA/MAA/BDDA) or poly(MMA/MAA/BDDA).
 4. Thecomposition according to claim 1, wherein adjacent microgel particlesare covalently bound together by covalent cross-links formed by thereaction of vinyl-containing moieties grafted onto the surfaces of themicrogel particles.
 5. The composition according to claim 4, wherein thevinyl containing moiety is a group of the formula -L-B, wherein L is abond or linking group; and B is a group comprising a vinyl functionalgroup.
 6. The composition according to claim 5, wherein the vinyl moietyhas the formula:

wherein L is a bond or a linking group and R₁, R₂ and R₃ are selectedfrom H or (1-3C)alkyl.
 7. The composition according to claim 1, whereinadjacent microgel particles are bound together by a cross-linked polymernetwork that interpenetrates adjacent microgel particles and therebybinds the particles together, wherein the polymer network is formed bythe polymerisation of a water soluble cross-linking monomer comprisingtwo or more vinyl groups.
 8. The composition according to claim 7,wherein the cross-linking monomer has the following formula:

wherein: (a) R²¹, R²², R²³, R³¹, R³² and R³³ may be independentlyselected from a group consisting of H; CH₃; a linear or branched alkylgroup; or a N-alkyl group of up to 10 C units; and wherein (b) R²⁴ maybe independently selected from a group consisting of: (i)—C(═O)—O—R³⁴—O—C(═O)—, wherein R³⁴ may comprise —CH₂—, —CH₂CH₂— or alinear or branched alkyl group, such as a methylene chain, which may beup to 20 C chains in length; or —C₆H₄—; or C₆H₃R³⁵, wherein R³⁵comprises substituents such alkyl, for example, CH₃; a halogen group; oran amide group; or other di- or tri-substituted phenyl groups containingmore than one of these substitutents; (ii) —C(═O)—O—R³⁶—C(═O)—, whereinR³⁶ may be —(CH₂CH₂O)_(n)— wherein n may be from 1 to 30; (iii)—C(═O)—O—R³⁷R³⁸R³⁷—, wherein R³⁷ may comprise degradable ester linkages,for example lactone, —[(CH₂)₅C(═O)—O]_(m)—, lactide,—[CH(CH₃)C(═O)—O]_(m)—, glycolide, —[CH₂C(═O)—O]_(m)—, wherein m may befrom 1 to 50, and wherein R³⁸ may be —(CH₂CH₂O)_(n)—, wherein n may befrom 1 to 30; (iv) —C(═O)—O—R³⁹—, wherein R³⁹ may comprise degradableester linkages, for example lactone, [(CH₂)₅C(═O)—O]_(m)—, lactide,[CH(CH₃)C(═O)—O]_(m)—, glycolide, [CH₂C(═O)—O]_(m)—, wherein m isbetween 1 to 100; (v) allylacrylates, for example —C(═O)—O—R⁴⁰—, whereinR⁴⁰ may be —CH₂—, —CH₂CH₂— or a linear, or branched, methylene chain upto 20 C chains in length, or —C₆H₄—, C₆H₃R⁴¹, wherein R⁴¹ may comprisesubstituents, such as alkyl, CH₃, a halogen or an amide group or otherdi- or tri-substituted phenyl groups containing more than one of thesesubstitutents; (vi) vinylbenzenes, for example C₆H₄ or C₆H₃R⁴² whereinR⁴² comprises substituents, such as alkyl; CH₃; a halogen or an amidegroup (see (iii) above); or other substituted phenyl groups containingmore than one of these substitutents; (vii) acrylamides, for exampleC(═O)—NR⁴³—R⁴⁴—NR⁴⁵C(═O)—, wherein R⁴³ and R⁴⁴ may be independentlyselected from a group consisting of H; CH₃; a linear or branched alkylgroup; a dialkyl group; a N-alkylgroup, of up to 10 C units; and whereinR⁴⁴ may comprise —CH₂—, —CH₂CH₂— or a linear, or branched, methylenechain up to 20C chains in length; or —C₆H₄—, C₆H₃R⁴¹ wherein R⁴¹comprises substituents, such as alkyl; CH₃; a halogen or an amide groupor other di- or tri-substituted phenyl groups containing more than oneof these substitutents; (viii) trifunctional cross-linking monomers,wherein R²⁴ comprises any of the groups listed in (b), as well asR²¹R²²C═CR²³, where R²¹, R²² and R²³ are described in (a); (ix)tetrafunctional cross-linking monomers, wherein R²⁴ comprises any of thegroups listed in (b), well as R²¹R²²C═CR²³ and R³¹R³²C=CR³³, whereinR²¹, and R³³ are described in (a); and (x) wherein R²⁴ may contain anycombination of the groups listed in (b).
 9. The composition according toclaim 1, wherein adjacent microgel particles are bound together by both:(i) a covalent cross-links formed by the reaction of vinyl-containingmoieties grafted onto the surfaces of the microgel particles; and (ii) across-linked polymer network that interpenetrates adjacent microgelparticles and thereby binds the particles together, wherein the polymernetwork is formed by the polymerisation of a water soluble cross-linkingmonomer comprising two or more vinyl groups.
 10. The microgel particlecomprising a vinyl-containing moiety as defined in claim 4, grafted onto its surface.
 11. A process for the preparation of a compositionaccording to claim 4, the process comprising: (i) providing, in anaqueous medium, a plurality of microgel particles; and (ii) causing themicrogel particles to swell so that adjacent microgel particles arebrought into contact with one another and facilitating connectionsbetween the microgel particles.
 12. The process for the preparation of acomposition according to claim 11, wherein: microgel particles to swellin the presence of a water soluble cross-linking monomer comprising twoor more vinyl groups such that adjacent microgel particles are broughtinto contact with one another and facilitating the polymerisation of thecross-linking monomer to form a cross-linked polymer network thatinterpenetrates the particles and binds adjacent microgel particlestogether.
 13. The process for the preparation of a composition accordingto claim 11, wherein the: the microgel particles include functionalvinyl-containing moieties grafted onto the surfaces of the microgelparticles; and wherein said swelling causes cross-linking monomercomprising two or more vinyl groups such that adjacent microgelparticles are brought into contact with one another; and (iii) includingthe further step of facilitating both: (a) the free radical coupling ofthe vinyl groups to covalently bind adjacent microgel particlestogether; and (b) the polymerisation of the cross-linking monomer toform a cross-linked polymer network that interpenetrates the particlesand binds adjacent microgel particles together.
 14. (canceled)
 15. Theprocess according to claim 11, wherein the microgel particles are causedto swell by a change in pH.
 16. The process according to claim 11,wherein the free-radical coupling of vinyl groups/polymerization of thecross-linking monomer is facilitated by the addition of an initiatorand, optionally, an accelerator.
 17. A process of preparing a microgelparticle comprising a plurality of vinyl-containing moieties graftedonto the surface of the microgel particle, the process comprisingreacting a microgel particle with a compound of the formula:Z-L-B wherein L and B are as defined in claim 5, and Z is a reactivegroups adapted to react with function groups of the microgel particle.18. A method of treating a subject suffering from a conditioncharacterised by damaged or degenerated soft tissue, the methodcomprising administering to a subject in need of such treatment, atherapeutically effective amount of a composition as defined in claim 1.19. The method according to claim 18, wherein the composition is formedin situ by administering microgel particles together reactants tocross-link vinyl-containing moieties grafted onto the surfaces of themicrogel particles; and/or polymerise a water soluble cross-linkingmonomer comprising two or more vinyl groups.
 20. The composition asdefined in claim 1 for use in the treatment of damaged or degeneratedsoft tissue.
 21. The process according to claim 11, wherein the microgelparticles includes functional vinyl-containing moieties grafted onto thesurfaces of the microgel particles; and wherein the swelling step bringsadjacent microgel particles into contact with one another andfacilitating connections between the microgel particles the free radicalcoupling of the vinyl groups to covalently bind adjacent microgelparticles together.